Peptides whose uptake by cells is controllable

ABSTRACT

A generic structure for the peptides of the present invention includes A-X-B-C, where C is a cargo moiety, the B portion includes basic amino acids, X is a cleavable linker sequence, and the A portion includes acidic amino acids. The intact structure is not significantly taken up by cells; however, upon extracellular cleavage of X, the B-C portion is taken up, delivering the cargo to targeted cells. Cargo may be, for example, a contrast agent for diagnostic imaging, a chemotherapeutic drug, or a radiation-sensitizer for therapy. X may be cleaved extracellularly or intracellularly. The molecules of the present invention may be linear, cyclic, branched, or have a mixed structure.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent application Ser. No. 10/699,562, filed Oct. 31, 2003, from which priority is claimed under 35 U.S.C. §120, the disclosure of which application is hereby expressly incorporated by reference in its entirety.

STATEMENT OF FEDERALLY-SPONSORED RESEARCH

This work was supported in part by grants from the Department of Energy, DE-FG03-01ER63276 and from the National Institutes of Health Grants DK54441, GM54038 and NS27177 (NINCDS). The government may have certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention pertains to compositions and methods for transporting material across cell membranes, and methods for making such compositions.

2. Introduction

Cell membranes delimit the outer boundaries of cells, and regulate transport into and out of the cell interior. Made primarily of lipids and proteins, they provide a hydrophilic surface enclosing a hydrophobic interior across which materials must pass before entering a cell. Although many small, lipophilic compounds are able to cross cell membranes passively, most compounds, particles and materials must rely on active mechanisms in order to gain entry into a living cell.

Transmembrane Transport

Regulation of transport into and out of a cell is vital for its continued viability. For example, cell membranes contain ion channels, pumps, and exchangers capable of facilitating the transmembrane passage of many important substances. However, transmembrane transport is selective: in addition to facilitating the entry of desired substances into a cell, and facilitating the exit of others, a major role of a cell membrane is to prevent uncontrolled entry of substances into the cell interior. This barrier function of the cell membrane makes difficult the delivery of markers, drugs, nucleic acids, and other exogenous material into cells.

Over the last decade, peptide sequences that can readily enter a cell have been identified. For example, the Tat protein of the human immunodeficiency virus 1 (HIV-1) is able to enter cells from the extracellular environment (e.g., Fawell et al. P.N.A.S. 91:664-668 (1994)). A domain from Antennapedia homeobox protein is also able to enter cells (Vives, E., et al., J. Biol. Chem. 272, 16010-16017 (1997)): Such uptake is reviewed in, for example, Richard et al., J. Biol. Chem. 278(1):585-590 (2003).

Such molecules that are readily taken into cells may also be used to carry other molecules into cells along with them. Molecules that are capable of facilitating transport of substances into cells have been termed “cell-penetrating peptides” (CPPs), protein transduction domains, and “membrane translocation signals” (MTS) (see, e.g., Tung et al., Advanced Drug Delivery Reviews 55:281-294 (2003)). The most important MTS are rich in amino acids such as arginine with positively charged side chains. Molecules transported into cell by such cationic peptides may be termed “cargo” and may be reversibly or irreversibly linked to the cationic peptides. An example of a reversible linkage is found in Zhang et al., P.N.A.S. 95:9184-9189 (1994)).

MTS molecules are discussed in, for example, Wender et al., P.N.A.S. 97:13003-13008 (2000); Hallbrink et al., Biochim. Biophys. Acta 1515:101-109 (2001); Derossi et al., Trends in Cell Biology 8:84-87 (1998); Rothbard et al., J. Med. Chem. 45:3612-3618 (2002); Rothbard et al., Nature Medicine 6(11):1253-1247 (2000); Wadia et al., Curr. Opinion Biotech. 13:52-56 (2002); Futaki et al; Bioconj. Chem. 12:1005-1011 (2001); Rothbard et al., U.S. Pat. No. 6,306,993; Frankel et al., U.S. Pat. No. 6,316,003; Rothbard et al., U.S. Pat. No. 6,495,663; and Monahan et al., U.S. Pat. No. 6,630,351. All patents and publications, both supra and infra, are hereby incorporated by reference in their entirety.

The uptake facilitated by MTS molecules is typically without specificity, enhancing uptake into most or all cells. Thus, although MTS molecules are capable of entering cells, and may be capable of enhancing the transport of other molecules linked to MTS molecules into cells, control and regulation of such transport remains difficult. However, it would be desirable to have the ability to target the delivery of cargo to a type of cell, or to a tissue, or to a location or region within the body of an animal. Accordingly, there remains a need in the art to target, to control and to regulate the delivery of cargo molecules by MTS molecules.

SUMMARY OF THE INVENTION

Molecules, compositions and methods for controlled delivery of substances into cells by transport molecules are provided. Molecules having features of the invention include peptide portions linked by a cleavable linker portion which may be a peptide. The inventors have found that the cellular uptake of MTS molecules with multiple basic amino acids can be inhibited or prevented by the addition of a portion having multiple negative charges at physiological pH, such as a peptide portion having multiple acidic amino acids. Thus, an embodiment of the invention provides compounds including a peptide portion A of between about 2 to about 20 acidic amino acids linked by a cleavable linker X to a peptide portion B of between about 5 to about 20 basic amino acids, so that while the peptide portion A is linked to the peptide portion B, uptake of the molecule into cells is inhibited or prevented. An acidic portion A may include some amino acids that are not acidic amino acids, or other moieties as well; similarly, a basic portion B may include some amino acids that are not basic amino acids, or other moieties as well. The inhibition or prevention of uptake of a basic portion B by an acidic portion A is termed “veto” of uptake of B. After cleavage of linker X so that peptide portion A may separate from the peptide portion B, portion B is able to enter a cell, the veto due to portion A having been removed. A cleavable linker X is preferably cleavable under physiological conditions.

In a further embodiment, a cargo portion C including a cargo moiety may be attached to basic portion B for transport of a cargo portion C along with B into a cell. Thus, an embodiment of the invention provides compounds including a peptide portion A of between about 2 to about 20 acidic amino acids in sequence linked by a cleavable linker X to a peptide portion B of between about 5 to about 20 basic amino acids, the peptide portion B being covalently attached to a cargo portion C to form a structure B-C, effective that while the peptide portion A is linked to the portion B, uptake of the MTS compound into cells is inhibited or prevented. Acidic portion A is able to veto of uptake of B-C. Transport across a cell membrane of cargo portion C linked to portion B is also thus inhibited or prevented by acidic portion A. After cleavage of linker X so that peptide portion A may separate from the peptide portion B, cargo portion C linked to peptide portion B is able to enter a cell as the uptake veto due to peptide portion A has been removed. A cleavable linker X is preferably cleavable under physiological conditions, allowing transport of cargo portion C into living cells. Cargo portion C may also be cleavably attached to basic portion B so that cargo portion C may separate from portion B within a cell.

Thus, an embodiment of the invention provides molecules including a peptide portion A having multiple acidic amino acids, e.g., between about 2 to about 20, preferably between about 5 and 20 acidic amino acids, the peptide portion A being effective to prevent the uptake of an MTS molecule having a peptide portion B having multiple basic amino acids e.g., between about 5 to about 20, preferably between about 9 to about 16 basic amino acids. Peptide portion A is also thus effective to prevent the enhancement of transport of cargo C across a cell membrane by a peptide portion B having multiple basic amino acids. Cleavage of a peptide portion A from a molecule that has a peptide portion B is effective to restore the ability of the remaining portion of the molecule including the portion B to be taken up by a cell. Cleavage of a peptide portion A from a molecule that has a cargo portion C covalently attached to a peptide portion B to form a structure B-C is effective to restore the ability of the structure B-C to be taken up by a cell.

In one embodiment, a molecule for controllably transporting a cargo moiety across a cell membrane includes a molecule or material having the structure A-X-B-C, where C comprises a cargo moiety, B comprises a peptide portion having multiple basic amino acids (e.g., between about 5 to about 20, preferably between about 9 to about 16 basic amino acids), B and C being covalently linked, A comprises a peptide portion having multiple acidic amino acids (e.g., between about 2 to about 20, preferably between about 4 to about 20 acidic amino acids), and X comprises a cleavable linker joining A with B-C. When linked with B-C, peptide portion A is effective to prevent the enhancement of transport of cargo C across a cell membrane. When the cleavable linker X is cleaved, the peptide portion A is freed from the rest of the molecule, including being freed from portion B and cargo portion C. The cargo portion C remains linked to portion B after cleavage of the cleavable linker X. The portion B is effective to enhance transport of cargo portion C across a cell membrane in the absence of portion A.

In embodiments of the invention, including molecules having the schematic structure A-X-B and molecules having the schematic structure A-X-B-C, acidic amino acids of portion A are glutamate, aspartate, or phosphoserine. An acidic amino acid has a side chain with a negative charge at pH 6.0, and may be glutamic acid, aspartic acid, or other acidic amino acid An acidic portion A having multiple acidic amino acids may have between about 2 to about 20, or between about 5 to about 20, or preferably from about 5 to about 9 acidic amino acids. In preferred embodiments, portion A comprises 5 to 9 glutamates or aspartates, and may comprise 5 to 9 consecutive glutamates or aspartates. In embodiments, acidic amino acids of portion A are D amino acids. In preferred embodiments, acidic amino acids of portion A are either D-glutamate, D-aspartate, or both.

A basic amino acid has a side chain with a positive charge at pH 6.0, and may be arginine, histidine, lysine, or other basic amino acid. In embodiments of the invention, the basic amino acids of portion B are either arginine, lysine or histidine. A basic portion B having multiple basic amino acids may have between about 5 to about 20, or between about 9 to about 16 basic amino acids. In preferred embodiments, portion B comprises about 9 to about 16 arginines, and may comprise about 9 to about 16 consecutive arginines. In embodiments of the invention, the basic amino acids of portion B are D amino acids. In preferred embodiments, basic amino acids of portion B are either D-arginine, D-lysine, D-histidine, or combinations thereof.

A cargo moiety may be any molecule, material, substance, or construct that may be transported into a cell by linkage to a MTS. A cargo portion C may include one or more cargo moieties. A cargo moiety may be, for example, a fluorescent moiety, a fluorescence-quenching moiety, a radioactive moiety, a radiopaque moiety, a paramagnetic moiety, a nanoparticle, a vesicle, a molecular beacon, a marker, a marker enzyme (e.g., horse-radish peroxidase (HRP), beta-galactosidase, or other enzyme suitable for marking a cell), a contrast agent (e.g., for diagnostic imaging), a chemotherapeutic agent, a radiation-sensitizer (e.g., for radiation therapy), a peptide or protein that affects the cell cycle, a protein toxin, or other cargo suitable for transport into a cell. In some embodiments where C is a fluorescent moiety, a fluorescence-quenching moiety is attached to portion A effective to quench the fluorescence of the fluorescent moiety C before cleavage of the linker X, and removing the quenching of fluorescent moiety C after cleavage of linker X.

A cleavable linker X serves to connect an acidic portion A with a basic portion B. A cleavable linker X may include, for example, between about 2 to about 100 atoms, or between about 6 to about 30 atoms. Cleavable linker portion X may include amino acid residues, and may be a peptide linkage of between about 1 to about 30, or between about 2 to about 10 amino acid residues. A cleavable linker X suitable for the practice of the invention may be a flexible linker. In preferred embodiments, a cleavable linker X suitable for the practice of the invention is a flexible linker, and may be about 6 to about 24 atoms in length. In embodiments of the invention, X may include a peptide linkage. In some embodiments of the invention, a cleavable linker X includes an aminocaproic acid (also termed aminohexanoic acid) linkage.

A cleavable linker X may be configured for cleavage exterior to a cell. In preferred embodiments of the invention, a cleavable linker X may be configured to be cleaved in conditions associated with cell or tissue damage or disease. Such conditions include, for example, acidosis; the presence of intracellular enzymes (that are normally confined within cells), including necrotic conditions (e.g., cleaved by calpains or other proteases that spill out of necrotic cells); hypoxic conditions such as a reducing environment; thrombosis (e.g., a linker X may be cleavable by thrombin or by another enzyme associated with the blood clotting cascade); immune system activation (e.g., a linker X may be cleavable by action of an activated complement protein); or other condition associated with disease or injury.

For example, a cleavable linker X may be configured for cleavage by an enzyme, such as a matrix metalloprotease. Other enzymes which may cleave a cleavable linker include, for example, urokinase plasminogen activator (uPA), lysosomal enzymes, cathepsins, prostate-specific antigen, Herpes simplex virus protease, cytomegalovirus protease, thrombin, caspase, and interleukin 1β converting enzyme. In embodiments of the invention, cleavable linker X may include the amino acid sequence PLGLAG (SEQ ID NO:1) or may include the amino acid sequence EDDDDKA (SEQ ID NO:2). In other embodiments, a cleavable linker X may include a S-S linkage, or may include a transition metal complex that falls apart when the metal is reduced. A molecule embodying features of the invention may have multiple linkers X linking a plurality of portions A having acidic amino acids to a structure B-C.

In embodiments of the invention, peptide portion A is located at a terminus of a polypeptide chain comprising B-C, or comprises the amino terminus of a polypeptide chain comprising B-C. A may be linked near to or at the amino terminus of a polypeptide chain comprising B-C, or A may be linked near to or at the carboxy terminus of a polypeptide chain comprising B-C. The polypeptide chain B-C may have ends that may be termed a B-side terminus and a C-side terminus. A cleavable linker X may be disposed near or at the B-side terminus, or may be disposed near or at the C-side terminus. In further embodiments, a portion or portions may be linear or may be cyclic. In embodiments, a cyclic molecule having features of the invention may have a single linker X or may have multiple linkers X.

In further embodiments of the invention, compositions and solutions, including pharmaceutical compositions are provided which include compounds of the invention having peptides capable of controllable delivery of cargo into a cell and a suitable carrier. Methods for producing such peptides capable of controllable delivery of cargo into a cell, and pharmaceutical compositions containing them are also provided. It will be understood that, in embodiments of the invention, peptoids, carbamates, vinyl polymers, and other molecules, with a cleavable linkage between an acidic and a basic portion, may also be provided.

The molecules, compositions and methods embodying features of the invention provide the advantages of controlling the uptake of basic amino acid-containing molecules into cells, and of controlling the delivery of cargo into cells. Such controlled uptake and controlled delivery of cargo into cells may be useful, for example, in treatment of patients having diseased cells or tissues. For example, delivery of an imaging contrast agent or antiproliferative agent as cargo may be directed to cancer cells, and not to all cells in a patient, offering the advantage of targeted delivery to the diseased cells, in order to enable noninvasive imaging or increase the effectiveness and decrease possible side effects of the treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic representation of a MTS molecule having features of the invention comprising a basic portion B, a linker portion X, and an acidic portion A.

FIG. 1B is a schematic representation of a cyclic MTS molecule having features of the invention comprising a basic portion B, two linker portions X, and an acidic portion A.

FIG. 2A is a schematic representation of a MTS molecule having features of the invention comprising a cargo portion C, a basic portion B, a linker portion X, and an acidic portion A.

FIG. 2B is a schematic representation of a MTS molecule having features of the invention comprising a cargo portion C, a basic portion B, a linker portion X, and an acidic portion A, the linker portion X connecting to the cargo portion C.

FIG. 2C is a schematic representation of a MTS molecule having features of the invention comprising a cargo C linked to multiple copies of MTS molecules each comprising a basic portion B, a linker portion X, and an acidic portion A.

FIG. 2D is a schematic representation of a MTS molecule having features of the invention comprising a cargo portion C, a basic portion B, multiple (two) linker regions X, and an acidic portion A.

FIG. 2E is a schematic representation of a cyclic MTS molecule having features of the invention comprising a cargo portion C, a basic portion B, in which two linker regions X flank an acidic portion A.

FIG. 2F is a schematic representation of a MTS molecule having features of the invention comprising a fluorescent cargo portion C, a basic portion B, a linker region X, and an acidic portion A having a quencher Q attached.

FIG. 3 is a schematic representation of a MTS molecule having features of the invention in which a cargo portion C is a contrast agent or drug, a basic portion B is a sequence of eight to ten D-arginine residues, a linker portion X is a cleavable linker that may be cleaved by proteolytic enzymes or reducing environment found near cancerous cells, and an acidic portion A is an inhibitory domain comprising D-amino acids.

FIG. 4 is a schematic representation of a MTS molecule of FIG. 3 having features of the invention in which the cleavable linker is not cleaved near normal tissue, showing the inability of a molecule of FIG. 3 to facilitate the entry of cargo into normal tissue.

FIG. 5 is a schematic representation of a MTS molecule of FIG. 3 having features of the invention in which the cleavable linker is cleaved by proteolytic enzymes or by the reducing environment found near cancer cells, showing the ability of a molecule of FIG. 3 to facilitate cargo entry into diseased tissue.

FIG. 6A illustrates a High Pressure Liquid Chromatography (HPLC) chromatogram of a peptide having features of the invention before cleavage of linker portion X that is a substrate for enterokinase.

FIG. 6B illustrates a HPLC chromatogram of the peptide of FIG. 6A after cleavage of linker portion X by enterokinase.

FIG. 7A illustrates a HPLC chromatogram of a peptide having features of the invention before cleavage of linker portion X that is a substrate for matrix metalloproteinase-2 (MMP-2).

FIG. 7B illustrates a HPLC chromatogram of the peptide of FIG. 7A after cleavage of linker portion X by MMP-2.

FIG. 8 illustrates the mean fluorescence measured by Fluorescence-Activated Cell Sorter (FACS) analysis of Jurkat cell populations incubated for ten minutes with MTS molecules having features of the invention, with fluorescent cargo moieties.

FIG. 9 illustrates the mean fluorescence measured by FACS analysis of Jurkat cell populations incubated for ten minutes with MTS molecules having features of the invention, with fluorescent cargo moieties.

FIG. 10 illustrates the mean fluorescence measured by FACS analysis of Jurkat cell populations incubated for ten minutes with MTS molecules having features of the invention, with fluorescent cargo moieties.

FIG. 11 illustrates the mean fluorescence measured by FACS analysis of Jurkat cell populations incubated for ten minutes with MTS molecules having features of the invention, with fluorescent cargo moieties.

FIG. 12 illustrates the mean fluorescence measured in Jurkat cells incubated for one hour with the MTS molecules of FIG. 11.

FIG. 13 illustrates the mean fluorescence measured in Jurkat cells incubated for ten minutes with MTS molecules having a disulfide linker connecting an acidic portion with a fluorescently labeled basic portion, or with the fluorescently labeled basic portion alone.

FIG. 14 illustrates some moieties suitable as part or all of a cargo portion of an MTS molecules having features of the invention.

FIG. 15 illustrates some moieties suitable for use as part or all of an acidic portion A.

FIG. 16 illustrates some moieties suitable for use as part or all of a linker X.

FIG. 17 illustrates some moieties suitable for use as part or all of a basic portion B.

FIG. 18 illustrates some polymeric moieties suitable for use as part or all of an acidic portion A.

FIG. 19 is a schematic diagram of activatable cell-penetrating peptides (ACPPs). Cellular uptake induced by a cationic peptide is blocked by a short stretch of acidic residues attached by a cleavable linker. Once the linker is cleaved, the acidic inhibitory domain drifts away, and the cationic cell-penetrating peptide (CPP) is free to carry its cargo into cells.

FIG. 20 illustrates association of ACPPs with live HT-1080 cells depends on cleavage by MMP-2, as demonstrated by FACS analysis (A) and microscopy (B and C). (A) Trace 1 (blue) shows untreated cells. Traces 2 (orange) and 3 (green) show cells incubated for 10 min with 1 μM uncleaved or precleaved peptide, respectively. Cells incubated with 1 μM r₉k(Cy5) are shown in red (Trace 4). (B) HT-1080 cells were incubated with 1 μg/ml Hoechst 33258 (Left) and 1.25 uncleaved peptide (Center) and imaged at Hoechst or Cy5 wavelengths (overlaid at Right). (C) Results from a similar experiment with cleaved peptide. The arrowheads indicate possible nucleoli.

FIG. 21 Nuclear Overhauser effects observed in two-dimensional NMR of a simple ACPP, succinyl-e₈-XPLGLAG-r₉-Xk, where X denotes 6-aminohexanoyl. Dashed red lines indicate observed nuclear Overhauser effects, and the green line highlights the peptide outline for clarity.

FIG. 22 illustrates visualization of HT-1080 tumor xenografts with activatable CPPs. HT-1080 tumors were implanted into the mammary fat pad of nude mice and allowed to grow until they reached 5-7 mm in diameter. (A1) A live anesthetized animal imaged 50 min after injection with 6 nmol of cleavable peptide. (A2 and A3) Tumor and muscle histology from a different animal killed 30 min after injection. (B1-B3) A similar experiment with the scrambled peptide. (Scale bars, 30 μm.)

FIG. 23 illustrates standardized uptake values (SUV) of cleavable and non-cleavable peptides in various tissues in mice.

FIG. 24. NMR spectra of the H^(β/γ)(δ₁)-H^(N)(δ₂) region of the peptide. Evidence for cross-strand interactions between D-arg and D-glu residues. A. NOESY spectrum. Blue dashed line at 8.65 ppm corresponds to C. B. TOCSY spectrum. Blue dashed lines at 8.65 and 8.59 correspond to D and E, respectively. C. 1D NOESY vector at the H^(N) of a D-arg. D. 1D TOCSY vector at the H^(N) of a D-arg. E. 1D TOCSY vector at the H^(N) of a D-glu.

FIG. 25. NOESY spectrum of the H^(N)(δ₁)-H^(N)(δ₂) region indicates sequential H^(N)-H^(N) backbone interactions through the residues of the linker region. Symmetry related cross-peaks are labeled once at either side of the diagonal.

FIG. 26. TPEN inhibits staining of squamous cell carcinoma specimens by a cleavable ACPP, (5 kDa PEG)-eeeeeeeeeXPLGLAG-rrrrrrrrrXk(Cy5), where X denotes 6-aminohexanoyl (SEQ ID NO: 52). Tissue slices were stained with 1 μM ACPP in the absence (A) or presence (B) of 1 μM TPEN. The slice shown in (A) contains regions of tumor (top right) as well as normal tissue. The slice shown in (B) contains only tumor.

FIG. 27 illustrates cleavage kinetics for MMP-2 cleavage of H₂N-e₆-XPLGLAG-r₉-Xc(Cy5)-CONH₂. where X is aminohexanoic acid.

FIG. 28 illustrates the dependence of uptake on cleavage by matrix metalloprotease-2 (MMP-2).

FIG. 29 illustrates the dependence of cargo uptake on peptide cleavage.

FIG. 30 illustrates localization of MMP-2-positive tumors in nude mice by imaging of fluorescence from cleaved peptides.

FIG. 31 illustrates the dependence of observed fluorescence intensity on peptide cleavage in vivo.

FIG. 32 provides images of human HT1080 tumors xenografted into nude mice illustrating improved contrast with cleavable peptides.

FIG. 33 provides images of cleavable peptide-derived fluorescence in spontaneous mammary tumors in mice, and provides gel images showing presence of cleaved peptide in tumor.

FIG. 34 provides images of cleavable peptide-derived fluorescence in metastasis and surrounding macrophages in lymph nodes in mice.

FIG. 35 provides images of resected human squamous cell carcinoma, and shows that gelatinase activity is increased in tumor.

FIG. 36 provides images of resected human squamous cell carcinoma.

FIG. 37 illustrates a scheme for generating phage particles decorated with ACPPs suitable for directing phage to sites having enzymatic activity for cleavage and delivery of phage to target tissues and cells.

FIG. 38 illustrates a scheme for sequence-dependent phage accumulation in tumors, and presents data illustrating increased phage uptake in target tumors.

FIG. 39 illustrates a scheme for attachment of a radioactive compound to an ACPP for delivery of the radioactive moiety to a target cell.

FIG. 40 provides data demonstrating increased delivery of radioactive cargo to target cells with enzymatic cleavage.

FIG. 41 provides data demonstrating enzymatic cleavage-dependent increase in delivery of radioactive cargo to target cells.

FIG. 42 illustrates a scheme for providing an MMP substrate for enhancement of magnetic resonance imaging (MRI) images.

FIG. 43 illustrates increased uptake of MRI contrast agent upon enzymatic cleavage of ACPP including contrast agent.

FIG. 44 illustrates a cyclic ACPP.

FIG. 45 illustrates a scheme for synthesizing a cyclic ACPP. Polyethylene glycol (PEG) may be attached to the cyclic ACPP to provide a PEGylated cyclic ACPP.

FIG. 46 illustrates a synthesized cyclic ACPP cleavable by MMP, and a scheme for its modification by addition of PEG and of a fluorescent label.

FIG. 47 provides data demonstrating self-quenching by cyclic ACPPs prior to enzymatic cleavage.

FIG. 48 schematically illustrates cyclic ACPP peptides that require cleavage at two sites for activation.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, a generic structure for peptides having features of the invention is A-X-B, where peptide portion B includes between about 5 to about 20 basic amino acids, X is a cleavable linker portion, preferably cleavable under physiological conditions, and where peptide portion A includes between about 2 to about 20 acidic amino acids. In some embodiments of molecules having features of the invention, peptide portion B includes between about 5 to about 20, or between about 9 to about 16 basic amino acids, and may be a series of basic amino acids (e.g., arginines, histidines, lysines, or other basic amino acids). In some embodiments of molecules having features of the invention, peptide portion A includes between about 2 to about 20, or between about 5 to about 20 acidic amino acids, and may be series of acidic amino acids (e.g., glutamates and aspartates or other acidic amino acids). A schematic representation of a MTS molecule having features of the invention comprising a basic portion B, a linker portion X, and an acidic portion A is presented in FIG. 1A. In embodiments, MTS molecules having features of the invention may be cyclic molecules, as schematically illustrated in FIG. 1B. Thus, MTS molecules having features of the invention may be linear molecules, cyclic molecules, or may be linear molecules including a cyclic portion.

As discussed above, molecules including a multiple basic amino acids, such as a series of basic amino acids, are often taken up by cells. However, the present inventors have discovered that molecules having structures including a basic portion B, a linker portion X, and an acidic portion A are not taken up by cells. An acidic portion A may include amino acids that are not acidic. Acidic portion A may comprise other moieties, such as negatively charged moieties. In embodiments of MTS molecules having features of the invention, an acidic portion A may be a negatively charged portion, preferably having about 2 to about 20 negative charges at physiological pH, that does not include an amino acid. A basic portion B may include amino acids that are not basic. Basic portion B may comprise other moieties, such as positively charged moieties. In embodiments of MTS molecules having features of the invention, a basic portion B may be a positively charged portion, preferably having between about 5 and about 20 positive charges at physiological pH, that does not include an amino acid. Including an acidic portion A is effective to inhibit or prevent the uptake of a portion B into cells. Such a block of uptake that would otherwise be effected by the basic amino acids of portion B may be termed a “veto” of the uptake by the acidic portion A. The present inventors have made the further surprising discovery that cleavage of linker X, allowing the separation of portion A from portion B is effective to allow the uptake of portion B into cells.

In a further embodiment, a generic structure for peptides having features of the invention is A-X-B-C, where C is a cargo moiety, X a linker, A an acidic portion, and B a basic portion. An acidic portion A may include amino acids that are not acidic. Acidic portion A may comprise other moieties, such as negatively charged moieties. In embodiments of MTS molecules having features of the invention, an acidic portion A may be a negatively charged portion, preferably having about 2 to about 20 negative charges at physiological pH, that does not include an amino acid. A basic portion B may include amino acids that are not basic. Basic portion B may comprise other moieties, such as positively charged moieties. In embodiments of MTS molecules having features of the invention, a basic portion B may be a positively charged portion, preferably having between about 5 and about 20 positive charges at physiological pH, that does not include an amino acid. In preferred embodiments, the amount of negative charge in portion A is approximately the same as the amount of positive charge in portion B.

A cargo moiety C may be, for example, a contrast agent for diagnostic imaging, or a chemotherapeutic drug or radiation-sensitizer for therapy. B may be, for example, a peptide portion having between about 5 to about 20 basic amino acids, such as a series of basic amino acids (arginines are preferred, although histidines are also suitable, as are lysines or other basic amino acids). X is a cleavable linker that is preferably cleavable under physiological conditions. A may be a peptide portion having between about 2 to about 20 about 2 to about 20 acidic amino acids, such as a series of acidic amino acids. In some embodiments of molecules having features of the invention, glutamates and aspartates are preferred acidic amino acids for peptide portion A. A schematic representation of a MTS molecule having features of the invention comprising a cargo portion C, a basic portion B, a linker portion X, and an acidic portion A is presented in FIG. 2A.

The present inventors have made the surprising discovery that including an acidic portion A is also effective to inhibit or prevent the uptake into cells of molecules combining a portion B and a portion C. The present inventors have made the further discovery that cleavage of linker X, allowing the separation of portion A from portion B is effective to allow the uptake of portions B and C into cells. Thus, delivery of cargo C can be controlled and enhanced by molecules having features of the invention.

For example, when peptide portion A contains about 5 to about 9 consecutive glutamates or aspartates, and X is a flexible linker of about 2 to about 100, or about 6 to about 30 atoms in length, the normal ability of a peptide portion B (e.g., a sequence of nine consecutive arginine residues) to cause uptake into cells is blocked. Cleavage of linker X allows the separation of portion A from portion B and portion C, alleviating the veto by portion A. Thus, when separated from A, the normal ability of portion B to effect the uptake of cargo C into cells is regained. Such cellular uptake typically occurs near the location of the cleavage event. Thus, design of cleavable linker X such that it is cleaved at or near a target cell is effective to direct uptake of cargo C into target cells. Extracellular cleavage of X allows separation of A from the rest of the molecule to allow uptake into cells.

A MTS molecule having features of the invention may be of any length. In embodiments of MTS molecules having features of the invention, a MTS molecule may be about 7 to about 40 amino acids in length, not including the length of a linker X and a cargo portion C. In other embodiments, particularly where multiple non-acidic (in portion A) or non-basic (in portion B) amino acids are included in one or both of portions A and B, portions A and B of a MTS molecule may together be about 50, or about 60, or about 70 amino acids in length. A cyclic portion of an MTS may include about 12 to about 60 amino acids, not including the length of a linker X and a cargo portion C. For example, a linear MTS molecule having features of the invention may have a basic portion B having between about 5 to about 20 basic amino acids (preferably between about 9 to about 16 basic amino acids) and an acidic portion A having between about 2 to about 20 acidic amino acids (e.g., between about 5 to about 20, preferably between about 5 to about 9 acidic amino acids). In some preferred embodiments, a MTS molecule having features of the invention may have a basic portion B having between about 9 to about 16 basic amino acids and between about 5 to about 9 acidic amino acids.

In healthy cells, the intact compound of structure A-X-B or A-X-B-C would not be able to enter the cell because of the presence of portion A. Thus, a strictly intracellular process for cleaving X would be ineffective to cleave X in healthy cells since portion A, preventing uptake into cells, would not be effectively cleaved by intracellular enzymes in healthy cells since it would not be taken up and would not gain access to such intracellular enzymes. However, where a cell is injured or diseased, so that such intracellular enzymes leak out of the cell, cleavage of A would occur, allowing entry of portion B or B-C into the cell, effecting targeted delivery of portion B or of cargo portion C to neighboring cells.

Portions A and B may include either L-amino acids or D-amino acids. In embodiments of the invention, D-amino acids are preferred for the A and B portions in order to minimize immunogenicity and nonspecific cleavage by background peptidases or proteases. Cellular uptake of oligo-D-arginine sequences is known to be as good or better than that of oligo-L-arginines. The generic structures A-X-B and -A-X-B-C can be effective where A is at the amino terminus and where A is at the carboxy terminus, i.e. either orientation of the peptide bonds is permissible. However, in embodiments where X is a peptide cleavable by a protease, it may be preferable to join the C-terminus of X to the N-terminus of B, so that the new amino terminus created by cleavage of X contributes an additional positive charge that adds to the positive charges already present in B.

Cargo portion C may be attached to B in any location or orientation. A cargo portion C need not be located at an opposite end of portion B than a linker X. Any location of attachment of C to B is acceptable as long as that attachment remains after X is cleaved. For example, a cargo portion C may be attached to or near to an end of portion B with linker X attached to an opposite end of portion B as illustrated in FIGS. 2A and 2B. A cargo portion C may also be attached to or near to an end of portion B with linker X attached to or near to the same end of portion B. In some embodiments of the invention, a linker X may link to a cargo portion C which is linked to a basic portion B as illustrated in FIG. 2B. FIG. 2C is a schematic representation of a MTS molecule having features of the invention comprising a cargo portion C linked to multiple basic portions B, each of which basic portions B are linked to a linker portion X, and via the linker to an acidic portion A.

A linker X may be designed for cleavage in the presence of particular conditions or in a particular environment. In preferred embodiments, a linker X is cleavable under physiological conditions. Cleavage of such a linker X may, for example, be enhanced or may be effected by particular pathological signals or a particular environment related to cells in which cargo delivery is desired. The design of a linker X for cleavage by specific conditions, such as by a specific enzyme, allows the targeting of cellular uptake to a specific location where such conditions obtain. Thus, one important way that MTS molecules having features of the invention provide specific targeting of cellular uptake to desired cells, tissues, or regions is by the design of the linker portion X to be cleaved by conditions near such targeted cells, tissues, or regions. After cleavage of a linker X, the portions B-C of the molecule are then a simple conjugate of B and C, in some instances retaining a relatively small, inert stub remaining from a residual portion of linker X.

A linker portion X may be cleavable by conditions found in the extracellular environment, such as acidic conditions which may be found near cancerous cells and tissues or a reducing environment, as may be found near hypoxic or ischemic cells and tissues; by proteases or other enzymes found on the surface of cells or released near cells having a condition to be treated, such as diseased, apoptotic or necrotic cells and tissues; or by other conditions or factors. An acid-labile linker may be, for example, a cis-aconitic acid linker. Other examples of pH-sensitive linkages include acetals, ketals, activated amides such as amides of 2,3 dimethylmaleamic acid, vinyl ether, other activated ethers and esters such as enol or silyl ethers or esters, imines, iminiums, enamines, carbamates, hydrazones, and other linkages. A linker X may be an amino acid or a peptide. A peptide linker may be of any suitable length, such as, for example, about 3 to about 30, or preferably about 6 to about 24 atoms in sequence (e.g., a linear peptide about 1 to 10 or preferably about 2 to 8 amino acids long). A cleavable peptide linker may include an amino acid sequence recognized and cleaved by a protease, so that proteolytic action of the protease cleaves the linker X.

One important class of signals is the hydrolytic activity of matrix metalloproteinases (MMPs), which are very important in the invasive migration of metastatic tumor cells. MMPs are also believed to play major roles in inflammation and stroke. MMPs are reviewed in Visse et al., Circ. Res. 92:827-839 (2003). MMPs may be used to cleave a linker X and so to allow separation of acidic portion A from portions B and C, allowing cellular uptake of cargo C so that cellular uptake of C is triggered by action of MMPs. Such uptake is typically in the vicinity of the MMPs that trigger cleavage of X. Thus, uptake of molecules having features of the invention are able to direct cellular uptake of cargo C to specific cells, tissues, or regions having active MMPs in the extracellular environment.

For example, a linker X that includes the amino-acid sequence PLGLAG (SEQ ID NO: 1) may be cleaved by the metalloproteinase enzyme MMP-2 (a major MMP in cancer and inflammation). Cleavage of such a linker X occurs between the central G and L residues, causing cell uptake to increase by 10 to 20-fold (see Example 4). A great deal is known about the substrate preferences of different MMPs, so that linkers X may be designed that are able to bias X to be preferentially sensitive to particular subclasses of MMPs, or to individual members of the large MMP family of proteinases. For example, in some embodiments, linkers X designed to be cleaved by membrane-anchored MMPs are particularly preferred because their activity remains localized to the outer surface of the expressing cell. In alternative embodiments, linkers X designed to be cleaved by a soluble secreted MMP are preferred where diffusion of cargo C away from the exact location of cleavage may be desired, thereby increasing the spatial distribution of the cargo. Other linkers X cleavable by other MMPs are discussed in Example 9.

Hypoxia is an important pathological signal. For example, hypoxia is thought to cause cancer cells to become more resistant to radiation and chemotherapy, and also to initiate angiogenesis. A linker X suitable for cleavage in or near tissues suffering from hypoxia enables targeting of portion B and C to cancer cells and cancerous tissues, infarct regions, and other hypoxic regions. For example, a linker X that includes a disulfide bond is preferentially cleaved in hypoxic regions and so targets cargo delivery to cells in such a region. In a hypoxic environment in the presence of, for example, leaky or necrotic cells, free thiols and other reducing agents become available extracellularly, while the O₂ that normally keeps the extracellular environment oxidizing is by definition depleted. This shift in the redox balance should promote reduction and cleavage of a disulfide bond within a linker X. In addition to disulfide linkages which take advantage of thiol-disulfide equilibria, linkages including quinones that fall apart when reduced to hydroquinones may be used in a linker X designed to be cleaved in a hypoxic environment.

Necrosis often leads to release of enzymes or other cell contents that may be used to trigger cleavage of a linker X. A linker X designed for cleavage in regions of necrosis in the absence of hypoxia, for example, may be one that is cleaved by calpains or other proteases that may be released from necrotic cells. Such cleavage of linkers X by calpains would release the connected portions B-C from portion A, allowing cargo to be taken up by diseased cells and by neighboring cells that had not yet become fully leaky.

Acidosis is also commonly observed in sites of damaged or hypoxic tissue, due to the Warburg shift from oxidative phosphorylation to anaerobic glycolysis and lactic acid production. Such local acidity could be sensed either by making an acid-labile linker X (e.g., by including in X an acetal or vinyl ether linkage). Alternatively, or in addition, acidosis may be used as a trigger of cargo uptake by replacing some of the arginines within B by histidines, which only become cationic below pH 7.

Molecules having features of the invention are suitable for carrying different cargoes, including different types of cargoes and different species of the same types of cargo, for uptake into cells. For example, different types of cargo may include marker cargoes (e.g., fluorescent or radioactive label moieties) and therapeutic cargoes (e.g., chemotherapeutic molecules such as methotrexate or doxorubicin), or other cargoes. Where destruction of aberrant or diseased cells is therapeutically required, a therapeutic cargo may include a “cytotoxic agent,” i.e. a substance that inhibits or prevents the function of cells and/or causes destruction of cells. In some embodiments, a single molecule having features of the invention may include more than one cargo portion C so that a basic portion B may be linked to multiple cargoes C. Such multiple cargoes C may include marker cargoes, therapeutic cargoes, or other cargoes. Multiple cargo moieties may allow, for example, delivery of both a radioactive marker and an ultrasound or contrast agent, allowing imaging by different modalities. Alternatively, for example, delivery of radioactive cargo along with an anti-cancer agent, providing enhanced anticancer activity, or delivery of a radioactive cargo with a fluorescent cargo, allowing multiple means of localizing and identifying cells which have taken up cargo.

Delivery of cargo such as a fluorescent molecule may be used to visualize cells having a certain condition or cells in a region exhibiting a particular condition. For example, thrombosis (clot formation) may be visualized by designing a linker X to be cleaved by any of the many proteases in the blood clot formation cascade for delivery of a cargo including a fluorescent or other marker to the region. Similarly, complement activation may be visualized by designing a linker X to be cleaved by any one or more of the proteases in the complement activation cascades for delivery of a fluorescent or other marker to the region. Thus, fluorescent molecules are one example of a marker that may be delivered to target cells and regions upon release of a portion A upon cleavage of a linker X.

A molecule having features of the invention may include one or more linkers X so that an acidic portion A may be linked to portions B and C by one or more linkages. Such linkages connecting to portion A may be to portion B, to portion C, or to both portions B and C. Where a molecule having features of the invention includes multiple linkages X, separation of portion A from the other portions of the molecule requires cleavage of all linkages X. Cleavage of multiple linkers X may be simultaneous or sequential. Multiple linkages X may include linkages X having different specificities, so that separation of portion A from the other portions of the molecule requires that more than one condition or environment (“extracellular signals”) be encountered by the molecule. Cleavage of multiple linkers X thus serves as a detector of combinations of such extracellular signals. FIG. 2D shows a MTS molecule having features of the invention that includes two linker portions Xa and Xb connecting basic portion B with acidic portion A. FIG. 2E shows a cyclic MTS molecule having features of the invention that includes two linker regions Xa and Xb connecting basic portion B with acidic portion A. In the MTS molecules schematically illustrated in FIGS. 2D and 2E, both linkers Xa and Xb must be cleaved before acidic portion A is separated from basic portion B ailowing entry of portion B and cargo portion C (if any) to enter a cell. It will be understood that a linker region may link to either a basic portion B or a cargo portion C independently of another linker that may be present, and that, where desired, more than two linker regions X may be included.

Combinations of two or more linkers X may be used to further modulate the targeting and delivery of molecules to desired cells, tissue or regions. Boolean combinations of extracellular signals can be detected to widen or narrow the specificity of the cleavage of linkers X if desired. Where multiple linkers X are linked in parallel, the specificity of cleavage is narrowed, since each linker X must be cleaved before portion A may separate from the remainder of the molecule. Where multiple linkers X are linked in series, the specificity of cleavage is broadened, since cleavage on any one linker X allows separation of portion A from the remainder of the molecule. For example, in order to detect either a protease OR hypoxia (i.e., to cleave X in the presence of either protease or hypoxia), a linker X is designed to place the protease-sensitive and reduction-sensitive sites in tandem, so that cleavage of either would suffice to allow separation of the acidic portion A. Alternatively, in order to detect the presence of both a protease AND hypoxia (i.e., to cleave X in the presence of both protease and hypoxia but not in the presence of only one alone), a linker X is designed to place the protease sensitive site between at least one pair of cysteines that are disulfide-bonded to each other. In that case, both protease cleavage AND disulfide reduction are required in order to allow separation of portion A.

The fact that capillaries are often leaky around tumors and other trauma sites should enhance the ability of high molecular weight molecules (e.g., molecular weight of about 40 kDa or more) to reach the interstitial compartment. Since the cleavage of a linker X is typically extracellular, some bystander labeling is expected, i.e. cells that do not express the relevant protease but that are immediately adjacent to expressing cells are likely to pick up some of the cargo. For tumors, such bystander targeting is considered beneficial because of the heterogeneity of cell phenotypes and the wish to eliminate as high a percentage of suspicious cells.

The fact that a single mechanism can mediate uptake of both imaging and therapeutic cargoes will be particularly valuable, because imaging with noninjurious tracer quantities can be used to test whether a subsequent therapeutic dose is likely to concentrate correctly in the target tissue.

D amino acids may be used in MTS molecules having features of the invention. For example, some or all of the peptides of portions A and B may be D-amino acids in some preferred embodiments of the invention. In an embodiment of the invention suitable for delivering a detectable marker to a target cell, a MTS having features of the invention includes a contrast agent as cargo C attached to a basic portion B comprising 8 to 10 D-arginines. Acidic portion A may include D-amino acids as well. Similarly, a drug may be delivered to a cell by such molecules having a basic portion B including 8 to 10 D-arginines and an acidic portion A including acidic D-amino acids. A schematic representation of such MTS molecules is shown in FIG. 3.

It will be understood that a MTS molecule having features of the invention may include non-standard amino acids, such as, for example, hydroxylysine, desmosine, isodesmosine, or other non-standard amino acids. A MTS molecule having features of the invention may include modified amino acids, including post-translationally modified amino acids such as, for example, methylated amino acids (e.g., methyl histidine, methylated forms of lysine, etc.), acetylated amino acids, amidated amino acids, formylated amino acids, hydroxylated amino acids, phosphorylated amino acids, or other modified amino acids. A MTS molecule having features of the invention may also include peptide mimetic moieties, including portions linked by non-peptide bonds and amino acids linked by or to non-amino acid portions. For example, a MTS molecule having features of the invention may include peptoids, carbamates, vinyl polymers, or other molecules having non-peptide linkages but having an acidic portion cleavably linked to a basic portion having a cargo moiety.

The linker portion X may be designed so that it is cleaved, for example, by proteolytic enzymes or reducing environment, as may be found near cancerous cells. Such an environment, or such enzymes, are typically not found near normal cells. FIG. 4 illustrates a MTS molecule as shown in FIG. 3, having a cleavable linker X designed to be cleaved near cancerous cells. As illustrated in FIG. 4, the cleavable linker is not cleaved near normal tissue. FIG. 4 illustrates the ability of a MTS having a portion A capable of vetoing cellular uptake of a portion B, and of a portion B-C, blocking the entry of cargo into normal tissue.

However, as illustrated in FIG. 5, the linker portion X may be cleaved, for example, by proteolytic enzymes or reducing environment found near cancerous cells to deliver a marker or a drug to cancerous cells. As shown in FIG. 5, a MTS molecule of FIG. 3 with a cleavable linker X that is cleaved by proteolytic enzymes or by the reducing environment near cancer cells is able to facilitate cargo entry into diseased tissue. Thus, the selective cleavage of the linker X and the resulting separation of cargo C and basic portion B from acidic portion A allows the targeted uptake of cargo into cells having selected features (e.g., enzymes), or located near to, a particular environment. Thus, molecules having features of the invention are able to selectively deliver cargo to target cells without doing so to normal or otherwise non-targeted cells.

In some embodiments, cargo C may be a fluorescent molecule such as fluorescein. Fluorescent cargo moieties enable easy measurement by fluorescence microscopy or flow cytometry in unfixed cultured cells. However, oligoarginine sequences, such as make up portion B, have been demonstrated to import a very wide varieties of cargoes C, ranging from small polar molecules to nanoparticles and vesicles (Tung & Weissleder (2003) Advanced Drug Delivery Reviews 55: 281-294). Thus, in embodiments of the invention, a cargo portion C may be any suitable cargo moiety capable of being taken up by a cell while connected to a basic portion B.

For example, for in vivo imaging purposes, C may be labeled with a positron-emitting isotope (e.g. ¹⁸F) for positron emission tomography (PET), gamma-ray isotope (e.g. ^(99m)Tc) for single photon emission computed tomography (SPECT), a paramagnetic molecule or nanoparticle (e.g. Gd³⁺ chelate or coated magnetite nanoparticle) for magnetic resonance imaging (MRI), a near-infrared fluorophore for near-infra red (near-IR) imaging, a luciferase (firefly, bacterial, or coelenterate) or other luminescent molecule for bioluminescence imaging, or a perfluorocarbon-filled vesicle for ultrasound. For therapeutic purposes, for example, suitable classes of cargo include but are not limited to: a) chemotherapeutic agents such as doxorubicin, mitomycin, paclitaxel, nitrogen mustards, etoposide, camptothecin, 5-fluorouracil, etc.; b) radiation sensitizing agents such as porphyrins for photodynamic therapy, or ¹⁰B clusters or ¹⁵⁷Gd for neutron capture therapy; or c) peptides or proteins that modulate apoptosis, the cell cycle, or other crucial signaling cascades. Existing chemotherapeutic drugs may be used, although they may not be ideal, because they have already been selected for some ability to enter cells on their own. In embodiments of the molecules of the invention, cargoes that are unable to enter or leave cells without the help of the polybasic portion B may be preferred.

Cargo C may include a radioactive moiety, for example a radioactive isotope such as ²¹¹At, ¹³¹I, ¹²⁵I, ⁹⁰Y, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ²¹²Bi, ³²P, radioactive isotopes of Lu, and others.

Cargo portion C may include a fluorescent moiety, such as a fluorescent protein, peptide, or fluorescent dye molecule. Common classes of fluorescent dyes include, but are not limited to, xanthenes such as rhodamines, rhodols and fluoresceins, and their derivatives; bimanes; coumarins and their derivatives such as umbelliferone and aminomethyl coumarins; aromatic amines such as dansyl; squarate dyes; benzofurans; fluorescent cyanines; carbazoles; dicyanomethylene pyranes, polymethine, oxabenzanthrane, xanthene, pyrylium, carbostyl, perylene, acridone, quinacridone, rubrene, anthracene, coronene, phenanthrecene, pyrene, butadiene, stilbene, lanthanide metal chelate complexes, rare-earth metal chelate complexes, and derivatives of such dyes. Fluorescent dyes are discussed, for example, in U.S. Pat. No. 4,452,720, U.S. Pat. No. 5,227,487, and U.S. Pat. No. 5,543,295.

A cargo portion C may include a fluorescein dye. Typical fluorescein dyes include, but are not limited to, 5-carboxyfluorescein, fluorescein-5-isothiocyanate and 6-carboxyfluorescein; examples of other fluorescein dyes can be found, for example, in U.S. Pat. No. 6,008,379, U.S. Pat. No. 5,750,409, U.S. Pat. No. 5,066,580, and U.S. Pat. No. 4,439,356. A cargo portion C may include a rhodamine dye, such as, for example, tetramethylrhodamine-6-isothiocyanate, 5-carboxytetramethylrhodamine, 5-carboxy rhodol derivatives, tetramethyl and tetraethyl rhodamine, diphenyldimethyl and diphenyldiethyl rhodamine, dinaphthyl rhodamine, rhodamine 101 sulfonyl chloride (sold under the tradename of TEXAS RED®), and other rhodamine dyes. Other rhodamine dyes can be found, for example, in U.S. Pat. No. 6,080,852, U.S. Pat. No. 6,025,505, U.S. Pat. No. 5,936,087, U.S. Pat. No. 5,750,409. A cargo portion C may include a cyanine dye, such as, for example, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy 7.

Some of the above compounds or their derivatives will produce phosphorescence in addition to fluorescence, or will only phosphoresce. Some phosphorescent compounds include porphyrins, phthalocyanines, polyaromatic compounds such as pyrenes, anthracenes and acenaphthenes, and so forth, and may be, or may be included in, a cargo portion C. A cargo portion C may also be or include a fluorescence quencher, such as, for example, a (4-dimethylamino-phenylazo)benzoic acid (DABCYL) group.

A pair of compounds may be connected to form a molecular beacon, having complementary regions with a fluorophore and a fluorescent quencher associated together so that the fluorescence of the fluorophore is quenched by the quencher. One or both of the complementary regions may be part of the cargo portion C. Where only one of the complementary regions (e.g., the fluorescent moiety) is part of the cargo portion C, and where the quencher moiety is part of the linker X or the acidic portion A, then cleavage of the linker X will allow fluorescence of the fluorescent portion and detection of the cleavage. Upon cellular uptake, the fluorescent portion of a molecular beacon will allow detection of the cell. For example, as illustrated in FIG. 2F, a quencher Q may be attached to an acidic portion A to form a MTS molecule having features of the invention Q -A-X-B-C where cargo C is fluorescent and is quenched by Q. The quenching of C by Q is relieved upon cleavage of X, allowing fluorescent marking of a cell taking up portion B-C. The combination of fluorescence dequenching and selective uptake should increase contrast between tissues able to cleave X compared to those that cannot cleave X.

Cargo C may include a chemotherapeutic moiety, such as a chemical compound useful in the treatment of cancer, or other therapeutic moiety, such as an agent useful in the treatment of ischemic tissue, or of necrotic tissue, or other therapeutic agent.

MTS molecules having features of the invention may be synthesized by standard synthetic techniques, such as, for example, solid phase synthesis including solid phase peptide synthesis. An example of peptide synthesis using Fmoc is given as Example 1 below. For example, conventional solid phase methods for synthesizing peptides may start with N-alpha-protected amino acid anhydrides that are prepared in crystallized form or prepared freshly in solution, and are used for successive amino acid addition at the N-terminus. At each residue addition, the growing peptide (on a solid support) is acid treated to remove the N-alpha-protective group, washed several times to remove residual acid and to promote accessibility of the peptide terminus to the reaction medium. The peptide is then reacted with an activated N-protected amino acid symmetrical anhydride, and the solid support is washed. At each residue-addition step, the amino acid addition reaction may be repeated for a total of two or three separate addition reactions, to increase the percent of growing peptide molecules which are reacted. Typically, 1 to 2 reaction cycles are used for the first twelve residue additions, and 2 to 3 reaction cycles for the remaining residues.

After completing the growing peptide chains, the protected peptide resin is treated with a strong acid such as liquid hydrofluoric acid or trifluoroacetic acid to deblock and release the peptides from the support. For preparing an amidated peptide, the resin support used in the synthesis is selected to supply a C-terminal amide, after peptide cleavage from the resin. After removal of the strong acid, the peptide may be extracted into 1M acetic acid solution and lyophilized. The peptide can be isolated by an initial separation by gel filtration, to remove peptide dimers and higher molecular weight polymers, and also to remove undesired salts The partially purified peptide may be further purified by preparative HPLC chromatography, and the purity and identity of the peptide confirmed by amino acid composition analysis, mass spectrometry and by analytical HPLC (e.g., in two different solvent systems).

The invention also provides polynucleotides encoding MTS molecules described herein. The term “polynucleotide” refers to a polymeric form of nucleotides of at least 10 bases in length. The nucleotides can be ribonucleotides, deoxynucleotides, or modified forms of either type of nucleotide. The term includes single and double stranded forms of DNA. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, e.g., an expression vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA) independent of other sequences.

These polynucleotides include DNA, cDNA, and RNA sequences which encode MTS molecules having features of the invention, or portions thereof. Peptide portions may be produced by recombinant means, including synthesis by polynucleotides encoding the desired amino acid sequence. Such polynucleotides may also include promoter and other sequences, and may be incorporated into a vector for transfection (which may be stable or transient) in a host cell.

The construction of expression vectors and the expression of genes in transfected cells involves the use of molecular cloning techniques that are well known in the art. See, for example, Sambrook et al., Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989) and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., (Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., most recent Supplement). Nucleic acids used to transfect cells with sequences coding for expression of the polypeptide of interest generally will be in the form of an expression vector including expression control sequences operatively linked to a nucleotide sequence coding for expression of the polypeptide. As used herein, “operatively linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. A control sequence operatively linked to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the control sequences. “Control sequence” refers to polynucleotide sequences which are necessary to effect the expression of coding and non-coding sequences to which they are ligated. Control sequences generally include promoter, ribosomal binding site, and transcription termination sequence. The term “control sequences” is intended to include, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. As used herein, the term “nucleotide sequence coding for expression of” a polypeptide refers to a sequence that, upon transcription and translation of mRNA, produces the polypeptide. This can include sequences containing, e.g., introns. As used herein, the term “expression control sequences” refers to nucleic acid sequences that regulate the expression of a nucleic acid sequence to which it is operatively linked. Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence. Thus, expression control sequences can include appropriate promoters, enhancers, transcription terminators, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signals for introns, maintenance of the correct reading frame of that gene to permit proper translation of the mRNA, and stop codons.

Methods which are well known to those skilled in the art can be used to construct expression vectors containing the fluorescent indicator coding sequence and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination. (See, for example, the techniques described in Maniatis, et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y., 1989). Transformation of a host cell with recombinant DNA may be carried out by conventional techniques as are well known to those skilled in the art. Where the host is prokaryotic, such as E. coli, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl₂ method by procedures well known in the art. Alternatively, MgCl₂ or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell or by electroporation.

When the host is a eukaryote, such methods of transfection of DNA as calcium phosphate co-precipitates, conventional mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes, or virus vectors may be used. Eukaryotic cells can also be cotransfected with DNA sequences encoding the fusion polypeptide of the invention, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene. Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein. (Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982). Techniques for the isolation and purification of polypeptides of the invention expressed in prokaryotes or eukaryotes may be by any conventional means such as, for example, preparative chromatographic separations and immunological separations such as those involving the use of monoclonal or polyclonal antibodies or antigen.

It will be understood that the compounds of the present invention can be formulated in pharmaceutically useful compositions. Such pharmaceutical compositions may be prepared according to known methods. For example, MTS compounds having features of the invention, and having a cargo portion C that is, for example, a therapeutic moiety, may be combined in admixture with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and their formulation, inclusive of other human proteins, e.g. human serum albumin are described, for example, in Remington's Pharmaceutical Sciences by E. W. Martin, which is hereby incorporated by reference. Such compositions will contain an effective amount of the compounds hereof together with a suitable amount of vehicle in order to prepare pharmaceutically acceptable compositions suitable for effective administration. Dosages and dosing regimens may be determined for the indications and compounds by methods known in the art, including determining (e.g., in experimental animals) the effective dose which causes half of those treated to respond to the treatment (ED ₅₀) by providing a range of doses to experimental animals or subjects and noting the responses.

Example 1

Peptide Synthesis

A number of peptides whose cell uptake could be modulated were synthesized. In the following, the following symbols, where used, are used with the indicated meanings: Fl=fluorescein; aca=aminocaproic acid linker (—HN—(CH₂)₅—CO—), C=L-cysteine, E=L-glutamate, R=L-arginine, D=L-aspartate, K=L-lysine, A=L-alanine, r=D-arginine, c=D-cysteine, e=D-glutamate, P=L-proline, L=L-leucine, G=glycine, V=valine, I=isoleucine, M=methionine, F=phenylalanine, Y=tyrosine, W=tryptophan, H=histidine, Q=glutamine, N=arginine, S=serine, and T=threonine. In sequences discussed below, lower case letters indicate the D isomer of the amino acid.

Peptides were synthesized on a peptide synthesizer (Pioneer Peptide Synthesis System by Applied Biosystems) using solid phase synthesis method and commercial available Fmoc amino acids, resins, and the other reagents. The peptides were cleaved with TFA/thioanisole/triisopropylsilane or TFA/thioanisole/triisopropylsilane/ethanedithiol. Peptides were labeled with 5- (and -6)carboxyfluorescein succinimidyl ester on the amino group on the peptide or with 5-iodoacetamidofluorescein on the thiol group on the peptide. The crude peptide was purified on HPLC and lyophilized overnight. Each peptide composition was confirmed by mass spectrometry.

Example 2

Peptide Cleavage by Enterokinase

10 μl 0.38 mM peptide dissolved in water stock solution was added to 10 μl 1 U/μl Enterokinase (Invitrogen, EKmax) and the cleavage progress was monitored by injecting 5 μl of the reaction mixture on HPLC monitored at 440 nm. The peptide was designed to be a substrate for enterokinase, with cleavage by enterokinase expected between the K and A residues. A High Performance Liquid Chromatography (HPLC) chromatogram of the peptide EDDDDKA-aca-R₉-aca-C(Fl)-CONH₂ (SEQ ID NO: 3) (before cleavage of linker portion between K and A) is illustrated in FIG. 6A. (The term “R₉” indicates a sequence of nine arginines.) The HPLC chromatograms showed that the peptide was cleaved almost completely after 15 min reaction time. FIG. 6B illustrates the HPLC chromatogram of the peptide of FIG. 6A after cleavage by enterokinase. The new peak was collected and determined on a mass spectrometer. The determined mass corresponded (as expected) to cleavage between K and A in the sequence of EDDDDKA-aca-R₉-aca-C(Fl)-CONH₂. (SEQ ID NO: 3)

Example 3

Peptides Having Acidic Portions to Veto Uptake

Peptide molecules having features of the invention, having fluorescent cargo moieties connected to basic portions (having multiple arginine residues), these latter being linked by cleavable linkers to an acidic portion (having multiple glutamate residues), were synthesized and tested for ability to deliver cargo into cells. Peptides showing ability of oligoglutamates to veto oligoarginine-mediated cellular uptake include:

(SEQ ID NO: 5) F1-aca-CRRRRRRRRR-aca-EEEEEEEEEC-CONH₂ (linear or cyclic, 5-47) (SEQ ID NO: 6) F1-aca-CEEEE-aca-RRRRRRRRRC-CONH₂ (linear or cyclic, 6-10)

Peptides showing cleavage-dependent uptake include:

(SEQ ID NO: 7) H₂N-EEEEEDDDDKA-aca-RRRRRRRRR-aca-C(F1)-CONH₂ (6-14, Enterokinase substrate, cleaved after DDDDK) (SEQ ID NO: 8) H₂N-EDDDDKA-aca-RRRRRRRRR-aca-C(F1)-CONH2 (6-16, Enterokinase substrate) (SEQ ID NO: 9) H₂N-EEEEEDDDDKARRRRRRRRR-aca-C(F1)-CONH₂ (6-27, Enterokinase substrate) (SEQ ID NO: 10) H₂N-EEDDDDKA-aca-numu-aca-C(F1)-CON-H₂ (6-29, Enterokinase substrate) (SEQ ID NO: 11) H₂N-DDDDDDKARRRRRRRRR-aca-C(F1)-CONH₂ (7-2, Enterokinase substrate) (SEQ ID NO: 12) H₂N-EEDDDDKAR-aca-RR-aca-RR-aca-RR-aca-RR- aca-C(F1)-CONH₂ (7-4, Enterokinase substrate) (SEQ ID NO: 13) H2N-eeeeee-aca-PLGLAG-rrrrrrrrr-aca-c(F1)-CONH₂ (7-6, MMP-2 substrate, cleaved between PLG and LAG)

Example 4

Peptide Cleaved by Matrix Metalloproteinase-2 (MMP-2):

MMP-2 (5 μg in 88 μl) was activated from human rheumatoid synovial fibroblast proenzyme (Invitrogen) in Tris-HCl buffer as described by Stricklin et al (1983) Biochemist 22: 61 and Marcy et al (1991) Biochemistry 30: 6476), then incubated with 32 μl 0.5 mM peptide stock solution for one hour at room temperature. FIG. 7A illustrates a HPLC chromatogram of the substrate peptide before cleavage by MMP-2. Enzyme cleavage progress was monitored by HPLC at 215 nm absorbance. FIG. 7B is a HPLC chromatogram of the peptide after cleavage by MMP-2, showing complete conversion to a new species.

Example 5

FACS Analysis of Cell Uptake:

The human T cell line-wide type Jurkat cells were cultured in RPMI 1640 media with 10% (v/v) deactivated fetal calf serum (FBS) and reached density ˜1×10⁶ cells/ml. The media was refreshed one day before being used. Before the experiment, the Jurkat cells were washed with HBSS buffer three times and resuspended in HBSS at (0.5-1)×10⁶ cells/ml density. In the cell uptake experiment, cells were stained with 1 μM peptide or compound at room temperature for 10 mm, then washed twice with cold HBSS and submitted for FACS analysis. Cell uptake was monitored by fluorescence at 530 nm run on FACS and 5,000-10,000 events were recorded from cells judged to be healthy by their forward and side scatter. The data represent mean fluorescence of the recorded cell population indicating uptake of the fluorescently labeled compounds. In most experiments, FI-GGR₁₀—CONH₂ (abbreviated as “R10” on the graphs; SEQ ID NO: 49) was included as a positive control for uptake.

The mean fluorescence measured in Jurkat cells incubated for ten minutes with the indicated peptides (each with fluorescent cargo moieties) is shown in FIGS. 8, 9 and 10.

As shown in FIG. 9, compounds 6-14 (SEQ ID NO: 7) and 6-16 (SEQ ID NO: 8) showed greatly enhanced fluorescence, indicating much greater uptake, of the cleaved form of the peptides than the intact peptides. Similarly, as shown in FIG. 10, compounds 7-2 (SEQ ID NO: 11) and 7-6 (SEQ ID NO: 13) also showed greatly enhanced fluorescence after cleavage compared with the fluorescence of the uncleaved compounds. Thus, these results demonstrate prevention of cellular uptake of compounds having basic amino acids by linkage to an acidic portion. Additionally, these results demonstrate enhanced cellular uptake of fluorescent portions of these peptides (having basic amino acids) following cleavage of the acidic portions.

Such cellular uptake increases as incubation time increases. FIG. 11 illustrates the mean fluorescence measured in Jurkat cells incubated for ten minutes with the indicated peptides having fluorescent cargo moieties, basic and acidic portions, and cleavable linker portions. As shown in FIG. 12, the mean fluorescence measured in Jurkat cells incubated for one hour was increased compared to the fluorescence measured as shown in FIG. 11.

The ability of MTS molecules having disulfide linkers X to provide controlled delivery of a cargo portion was tested using peptide 7-45 (SEQ ID NO: 14) having the structure

in which a disulfide bond between the two cysteines links the acidic portion H₂N-eeeeeec-CONH₂ (SEQ ID NO: 15) with the basic portion Fl-rrrrrrrrrc-CONH₂ (SEQ ID NO: 16). The basic portion carries the cargo portion, fluorescent moiety Fl (fluorescein). As illustrated in FIG. 13, the mean fluorescence measured in Jurkat cells incubated for ten minutes with the intact 7-45 peptide (SEQ ID NO: 14) showed only a small amount of fluorescence above that of the background measured from the Jurkat cells alone. However, when the peptide was reduced with 25 mM tris(carboxyethyl)phosphine and 250 mM 2-mercaptoethanesulfonate for 15 min, which cleave the disulfide linker X, then incubated with Jurkat cells for ten minutes, the fluorescence taken up by the cells was comparable to that of cells incubated for 10 minutes in the presence of R10. Thus, a MTS molecule having features of the invention, with a disulfide linker X, is able to provide controlled delivery of cargo portion to cells.

Example 6

MTS Molecules Having Varying Lengths

MTS molecules having features of the invention may have different numbers of basic amino acids, different numbers of acidic amino acids, and different linkers. Several examples of different MTS molecules illustrating features of the invention are presented in this Example, in which a fluorescent cargo moiety is exemplified by fluorescein (Fl), a radioactive cargo moiety is exemplified by ¹²⁵I, and a therapeutic cargo by doxorubicin (DOX).

(SEQ ID NO: 17): EDA-aca-R₅-aca-C(F1)-CONH₂ (SEQ ID NO: 18) EDDDDKA-aca-R₆-aca-C(DOX)-CONH₂ (SEQ ID NO: 19) EEEDDDEEEDA-aca-R₉-aca-Y(¹²⁵I)-CONH₂ (SEQ ID NO: 20) ededdAAeeeDDDDKA-aca-R₁₁-aca-C(F1)-CONH₂ (SEQ ID NO: 21) eddedededDDDDKA-aca-R₆-AGA-R₆-aca-C(DOX)-CONH₂ (SEQ ID NO: 22) Ggedgddeeeeeeddeed-aca-PLGLAG-aca-R₈-AAA-R₁₂-aca- C(F1)-CONH₂ (SEQ ID NO: 23) eeddeeddKA-aca-R₇-aca-C(F1)-CONH₂ (SEQ ID NO: 24) eDDDDKA-aca-RGRGRRRAca-C(F1)-CONH₂ (SEQ ID NO: 25) eddddeeeeeee-aca-PLGLAGKA-aca-R₁₀-aca-C(F1)-CONH₂ (SEQ ID NO: 26) eeeeeeeeeeeeeeee-aca-DDDDKA-aca-R₂₀-aca-C(F1)- CONH₂ (SEQ ID NO: 27) eeeeeeeeeddddd-aca-DDDDKA-aca-R₁₇-aca-Y(¹²⁵I)- CONH₂ (SEQ ID NO: 28) dddddddddddddddd-aca-PLGLAG-aca-R₁₄-aca-C(DOX)- CONH₂

Example 7

Examples of Molecules Suitable for Use as Cargo Moieites

Examples of molecules suitable for attachment as cargo moieties to a basic portion B of a MTS molecule having features of the invention are illustrated in FIG. 14. The different exemplary molecules shown in FIG. 14 are each labeled by an identifier letter in parentheses. The molecules are shown having one bond that ends in a dot; the bond ending in a dot may be used to attach the cargo molecule to a basic portion B. A letter in brackets near the dotted bond indicates a suitable atom to which the cargo molecule might bind; for example, [N] indicates that the cargo molecule may bind to a nitrogen, such as a nitrogen of a lysine epsilon amino group, or a nitrogen of an alpha amino group of a peptide backbone of the MTS molecule. An [S] indicates a linkage to a sulfur atom, such as a cysteine sulfur atom.

More than one of these exemplary cargo molecules may be attached to a basic portion B, and basic portions B carrying multiple cargo molecules may have more than one type of cargo molecule attached. The cargo molecules may form part of more complex structures as well. For example, the dark circle in the cargo moiety labeled (k) represents a particle including a superparamagnetic iron oxide core, jacketed by crosslinked, aminated dextran (such particles typically have a radius of about 22 nanometers). Although only one pendant group is shown, such particles may have multiple pendant groups (typically about 4 to about 20).

Example 8

Examples of Acidic Moieties Suitable for Inclusion in an Acidic Portion A

An acidic portion A may include acidic moieties such as those illustrated in FIG. 15. Such moieties may be linked to a linker X and an acidic portion A by peptide bonds, disulfide bonds, or other bonds. A dashed line in the illustration indicates a possible attachment point. In this and subsequent figures, a moiety in brackets indicates a motif that may be repeated, with a letter (e.g., “x”) indicating the number of times that the motif may be repeated (which may take on a number of possible values, typically between about 1 and about 100, preferably between about 1 and about 20). It will be understood that such acidic moieties may be attached to an acidic portion A in any suitable manner. In embodiments, an acidic portion A of a MTS molecule having features of the invention may be partly comprised of, or mainly comprised of, or essentially completely comprised of acidic moieties such as those illustrated in FIG. 15.

Example 9

Examples of Linker Moieties

Linkers suitable for use in a MTS molecule having features of the invention may be peptides or other molecules cleavable by enzymes under physiological conditions. For example, linkers may be cleavable by such enzymes as metalloproteases. Linkers cleavable by MMP-2 have been discussed supra. In addition, for example, linkers cleavable by other metalloproteases, such as MMP-9, MMP-11, and MMP-14 are also suitable. For example, peptide linker cleavable by MMP-9 may include the peptide sequence

PR(S/T)(L/I)(S/T) (SEQ ID NO: 29)

where the letters in parentheses indicate that either one of the indicated amino acids may be at that position in the sequence. A peptide linker cleavable by MMP-11 may include the peptide sequence

GGAANLVRGG (SEQ ID NO: 30)

and peptide linker cleavable by MMP-14 (MT1-MMP) may include the peptide sequence

SGRIGFLRTA. (SEQ ID NO: 31)

A peptide linker cleavable by urokinase plasminogen activator (uPA) may include the peptide sequence

SGRSA (SEQ ID NO: 32)

A peptide linker cleavable by lysosomal enzymes may include one of more of the peptide sequences

GFLG, (SEQ ID NO: 33) ALAL, (SEQ ID NO: 34) and FK.

A peptide linker may be cleavable by a cathepsin. For example, a linker cleavable by cathepsin B may include a KK or a RR sequence, or may include both, where the cleavage would typically occur between the lysines or arginines. A peptide linker cleavable by cathepsin D may include the peptide sequence

PIC(Et)F-F, (SEQ ID NO: 35)

where C(Et) indicates S-ethylcysteine (a cysteine with an ethyl group attached to the thiol) and the “-” indicates the typical cleavage site in this and subsequent sequences. A peptide linker cleavable by cathepsin K may include the peptide sequence

GGPRGLPG. (SEQ ID NO: 36)

A peptide linker cleavable by prostate-specific antigen may include the peptide sequence

HSSKLQ-. (SEQ ID NO: 37)

A peptide linker cleavable by Herpes simplex virus protease may include the peptide sequence

LVLA-SSSFGY. (SEQ ID NO: 38)

A peptide linker cleavable by HIV protease may include the peptide sequence

GVSQNY-PIVG. (SEQ ID NO: 39)

A peptide linker cleavable by Cytomegalovirus protease may include the peptide sequence

GVVQA-SCRLA (SEQ ID NO: 40)

A peptide linker cleavable by Thrombin may include the peptide sequence

f(Pip)R-S (SEQ ID NO: 41)

where “f” indicates D-phenylalanine and “Pip” indicates piperidine-2-carboxylic acid (pipecolinic acid, a proline analog having a six-membered ring).

A peptide linker cleavable by Caspase-3 may include the peptide sequence

DEVD-. (SEQ ID NO: 42)

A peptide linker cleavable by Interleukin 1β converting enzyme may include the peptide sequence

GWEHD-G. (SEQ ID NO: 43)

In addition, linkers suitable for use in a MTS molecule having features of the invention may be cleavable by agents other than proteases under physiological conditions. Linkers may also be non-peptide molecules. Some examples of enzymatically and non-enzymatically cleavable moieties suitable as linkers are illustrated in FIG. 16. Examples of different cleavable linkers are shown along with an indication of conditions which lead to cleavage. For example, cleavage of the linker labeled (a) may be accomplished by beta-lactamase. Cleavage of the linker labeled (b) may be accomplished by exposure to light, such as to a single photon of violet light or to two photons of infrared light. Cleavage of the linker labeled (c) may occur under reducing conditions. Cleavage of the linkers labeled (d) and (e) may occur in acidic conditions. Action of an esterase may cleave the linker labeled (f), and a phosphatase may cleave the linker labeled (g).

Example 10

Examples of Basic Moieties Suitable for Inclusion in a Basic Portion B

A basic portion B may include basic moieties such as those illustrated in FIG. 17. Such moieties B may be linked to a linker X, cargo C, or to another part of a basic portion B by peptide bonds, disulfide bonds, or other bonds. A dot indicates a possible attachment point, while a letter enclosed by brackets indicates a possible atom to which such an attachment may be made (e.g., [S] indicates that a bond, such as a diusulfide bond, may be made to a sulfur atom; a [N] indicates a bond to a nitrogen may be made). It will be understood that such basic moieties may be attached to a basic portion B or other portions of a MTS molecule in any suitable manner. For example, the “X” shown in compound (c) of FIG. 17 indicates attachment of a linker X to the side-chain of a D-lysine residue. The amino acid portion of compound (c) of FIG. 17 is SEQ ID NO: 44; the amino acid portion of compound (d) of FIG. 17 is SEQ ID NO: 45; the amino acid portion of compound (e) of FIG. 17 is SEQ ID NO: 46; and the amino acid portion of compound (f) of FIG. 17 is SEQ ID NO: 47. In embodiments, a basic portion B of a MTS molecule having features of the invention may be partly comprised of, or mainly comprised of, or essentially completely comprised of basic moieties such as those illustrated in FIG. 17.

It will be understood that some combinations of A and B may be more suitable than others. For example, it is preferred that the same backbone structure be present in both portions A and B in a MTS molecule having features of the invention, so that, for example, both A and B are peptides, or both A and B are peptoids, or both A and B are carbamates. It is also preferred that the absolute value of the net charge of one portion be similar, or the same as, the absolute value of the net charge of the other portion so that, for example, A has approximately the same number of negative charges as B has positive charges.

Example 11

Examples of Polymeric Acidic Portions

In another embodiment, an acidic portion A may include or be part of a polymer. In preferred embodiments, the polymer has an average molecular weight of about 50 kDa or above. Such high molecular weights reduce immunogenicity and improve pharmacodynamics by slowing excretion and lengthening the residence time in the bloodstream. Furthermore, polymers of such size benefit from “enhanced permeability and retention” (EPR) in tumors, whose capillaries are much leakier than normal tissue and whose lymphatic drainage is often impaired. These properties cause polymers to have higher ratios of concentrations in tumor vs. normal tissue than those of low-molecular-weight drugs. For recent discussions of the benefits of polymeric carriers, see Kopecek et al (2001) J. Controlled Release 74: 147-158; Luo & Prestwich (2002) Current Cancer Drug Targets 2: 209-226; Maeda et al (2003) International Immunopharmacology 3: 319-328; and Torchilin & Lukyanov (2003) Drug Discovery Today 8: 259-266. This EPR effect leading to enhancement of concentration in tumor tissue compared to normal tissue should further reinforce the tumor selectivity resulting from preferential cleavage of the linker X of MTS molecules having features of the invention by enzymes or under conditions found near tumors. Cleavage of X is effective to release basic portion B and cargo C attached to B from a polymeric acidic portion A, allowing the uptake of B and C into cells. In preferred embodiments, the polymer carries a sufficient number of negative charges to veto uptake of B and C while linker X is still intact. Examples of such polymers are shown in FIG. 18. The amino acid portion of compound (c) of FIG. 18 is SEQ ID NO: 48.

Example 12

Examples of Tumor Imaging

The methods, compositions and systems disclosed herein may be used for selectively delivering molecules to tumor cells. Cellular association of polyarginine based, cell-penetrating peptides (CPPs) is effectively blocked when they are fused to an inhibitory domain made up of negatively charged residues. In this example, such fusions are termed “activatable CPPs” (ACPPs) because cleavage of the linker between the polycationic and polyanionic domains, typically by a protease, releases the CPP portion and its attached cargo to bind to and enter cells. Association with cultured cells typically increases 10-fold or more upon linker cleavage. In mice xenografted with human tumor cells secreting matrix metalloproteinases 2 and 9, ACPPs bearing a far-red-fluorescent cargo show in vivo contrast ratios of 2-3 and a 3.1-fold increase in standard uptake value for tumors relative to contralateral normal tissue or control peptides with scrambled linkers. Ex vivo slices of freshly resected human squamous cell carcinomas give similar or better contrast ratios. Because CPPs are known to import a wide variety of nonoptical contrast and therapeutic agents, ACPPs offer a general strategy toward imaging and treating disease processes associated with linker-cleaving activities such as extracellular proteases. References cited in this example are indicated by reference number, with the full citation for each numbered reference provided at the end of the example.

Molecular imaging and therapy in patients would greatly benefit from generic, rational mechanisms to target contrast agents and therapeutic drugs to diseased tissues, especially tumors (1). Currently, the main strategies are based on antibodies against surface markers or ligands for receptors preferentially expressed in the target tissue (2). Although antibodies have occasionally been successful in targeting tumors (3), their irreducible bulk hinders penetration of solid tumors and excretion of unbound reagent (4), and elaborate reengineering is required to minimize immunogenicity (5, 6). A few small molecule ligands (2 kDa or less) for endogenous receptors have been preliminarily explored, but robust tumor specificity is rare or nonexistent (4). A fundamental limitation of simple antibody or ligand binding is the lack of amplification, where each target molecule (typically of low abundance) can bind at most one probe. Some amplification can be achieved by incorporating the probe into polymers or nanoparticles, but the increase in bulk worsens access to diseased tissue and removal from healthy organs. None of these approaches help get drugs across the plasma membrane into the cytoplasm and nucleus of diseased cells, the most desirable loci for modifying signal transduction or triggering cell death. Certain polycationic sequences [variously dubbed cell penetrating peptides (CPPs), membrane-translocating sequences (MTS), or protein transduction domains] can bring covalently attached payloads into mammalian cells without requiring specific receptors. CPPs were first discovered within a domain from Antennapedia homeobox protein and the tat protein from HIV-1 (7, 8). A variety of multicationic oligomers, including VP-22 and guanidinium-rich sequences, as simple as 6-12 consecutive arginines are now known to be equally or more effective (9-11). D-Amino acids are at least as good as natural L-amino acids and possibly better because the unnatural isomers resist proteolysis (10-12). Cargoes ranging in size from metal chelates and fluorescent dyes (13, 14) to iron oxide nanoparticles (15) and liposomes (16) can be imported, although the detailed mechanisms and subcellular localizations remain poorly understood and may differ, depending on cargo size, cell type, CPP sequence, and other experimental variables (17, 18). Initial attachment of the polycations to the cell surface is avid, rapid, and probably mediated by electrostatic attraction for anionic phospholipids and glycosaminoglycans. Much of the subsequent internalization probably occurs by endocytosis, because delivery of bioactive cargoes to the cytosol and nucleus can be enhanced by inclusion of sequences known for acidification-dependent disruption of endosomes (19, 20).

We now demonstrate a generic targeting mechanism based on selective local unleashing of CPPs, as schematized in FIG. 19. Cellular uptake of CPPs can be largely blocked by fusing them by means of cleavable linkers to polyanionic sequences, which neutralize the polycations by forming intramolecular hairpins of ≈2-3 kDa. We call such constructs activatable CPPs (ACPPs), because cleavage of the linkers dissociates the inhibitory polyanions, releasing the polycationic peptides and their cargo to attach to and enter cells. The mechanism (FIG. 19) is a flexible, modular, amplifying strategy to concentrate imaging and therapeutic agents on and within cells in the immediate vicinity of extracellular cleavage activities, such as matrix metalloproteinases (MMPs) in tumors. We chose MMP-2 and MMP-9 as our primary initial targets because they are the best characterized proteases overexpressed by tumors (21). Currently, at least 26 members of the MMP family have been identified. They play a crucial role in extracellular matrix degradation, tissue invasion, and metastasis (21-26).

Materials and Methods: Peptide Synthesis and Fluorophore Labeling. Peptides were synthesized on an automatic peptide synthesizer by using standard protocols for fluorenylmethoxycarbonyl solid-phase synthesis. Further details and further information regarding peptide synthesis, fluorophore labeling, and poly(ethylene glycol) (PEG) attachment (PEGylation) is presented in Example 13.

Peptide Cleaved by MMP-2 (PLGLAG). MMP-2 proenzyme (5 μg in 80 μl of 50 mM Tris_HCl buffer) was activated with 2.5 mM 4-aminophenylmercuric acetate at 37° C. for 2 h. Afterward, we added 32 μl of 0.5 mM peptide stock solution and incubated the mixture for 1 hr at room temperature. Enzyme cleavage progress was monitored by HPLC. The HPLC chromatograms showed that near complete cleavage was accomplished after 30 min of incubation. The new peak was collected, and its mass was determined by mass spectroscopy. The mass spectrum indicated that the enzyme was cut between glycine and leucine residues of the MMP-2 substrates as predicted, giving products such as NH2-eeeeee-ahx-PLG (SEQ ID NO: 50) and LAG-rrrrrrrrr-ahx-c(Fl)-CONH₂ (SEQ ID NO: 51), where “ahx” indicates aminohexanoic acid (also termed “aminocaproic acid”).

FACS Analysis and Microscopy. Jurkat cells were cultured in RPMI medium 1640 plus 10% (vol_vol) FBS to a density of 0.5-1×10⁶ cells per ml. The media was refreshed 1 day before the assay of ACPPs. Cells were washed with Hanks' balanced salt solution (HBSS) buffer three times, resuspended in HBSS at 0.5-1×10⁶ cells per ml, stained with 1 μM peptide in HBSS at room temperature for 10 min, washed three times with cold HBSS, and analyzed by flow cytometry at 530-nm emission for fluorescein labeled peptides or at 675 nm for Cy5-labeled peptides. We collected 10,000 events from cells judged to be healthy by their forward and side scatter. Peptide association with HT-1080 cells was similarly quantified by flow cytometry after release from adherence with trypsin. For microscopic imaging, HT-1080 cells grown to 70% confluency were washed with HBSS three times, stained with 1.25 μM peptide and 1 μg/ml Hoechst 33258 (a nuclear stain), rinsed twice, trypsinized, replated on polylysinecoated dishes, and imaged for Cy5 content (excitation, 625-645 nm; emission, 665-695 nm) and Hoechst 33258 (excitation, 375-385 nm; emission, 420-460 nm).

Xenografts in Mice. Nude mice (age, 4-6 weeks) were injected s.c. with ≈10⁶ HT-1080 cells. Once the tumors had reached 5-7 mm in size (typically 1-2 weeks later), animals were anesthetized with 100 mg/kg ketamine and 5 mg/kg midazolam), weighed, and injected with ≈100 μl of 60 μM peptide through the tail vein. Animals were then imaged at various times by using a Nikon f/1.2 camera lens in front of a cooled charge-coupled device camera (SenSys, Photometrics, Tucson, Ariz.). For longer lasting imaging studies, animals were allowed to wake up after 2 h of anesthesia and were reanesthetized at ≈4 and 6 hr for further data collection. Plasma half-lives were determined by the decrease in fluorescence intensity of ≈5-μl blood samples withdrawn periodically into heparinized capillaries. After imaging was ended, animals were killed with halothane, and organs of interest were harvested and weighed. For frozen sectioning, tissues were added to OCT cryopreservative and frozen on dry ice and hexane. Samples were stored at −80° C. and cut into 5-μm sections at −20° C. by using a cryotome. Cy5 fluorescence was imaged as described above. To measure standardized uptake values (SUVs), 30 mg of each tissue was added to 100 μl of a buffered 1% SDS mixture (pH 7.6) and protease inhibitor mixture (Roche Diagnostics). The tissue was then homogenized, heated to 70° C. for 15 min, microwaved for 15 sec, centrifuged at 20,500×g for 15 min, then imaged on the same system used for whole mice. Two sets of standards (liver and kidney) were used to calibrate fluorescence intensity in terms of peptide concentration. From this calibration, the quantity of peptide in 30 mg of tissue for each organ was calculated. SUVs were calculated as the molality of peptide in the tissue divided by the total injected dose as mol/kg of body weight.

Squamous Cell Carcinoma Specimens. Human squamous cell carcinoma specimens from planned resections of neoplasms were collected postoperatively according to a protocol with institutional review board approval. The specimens were in ice-cold normal saline for 30 min during transport back to the laboratory, where they were cut by hand to ≈1-mm-thick slices, added to 1 ml of 1 μM peptide for 15 min at room temperature, rinsed five times for 2 min in 1 ml of HBSS, cryosectioned, and imaged as described above.

Results

Until Cleaved Off, Polyanionic Sequences Inhibit Association of CPPs with Cells. Given that the initial binding of CPPs to cells is believed to be electrostatic, we asked whether association with cells could be prevented by appending polyanionic sequences to give the polycations intramolecular diversions. Fluorescently labeled peptides were synthesized with nine arginine residues fused by means of cleavable linkers to six to nine consecutive acidic residues, usually glutamate. We incubated these peptides, either intact or with linkers precleaved, with Jurkat lymphocytes or HT-1080 fibrosarcoma cells and assessed cell fluorescence by flow cytometry and fluorescence microscopy of the live unfixed cells after washing away unbound peptides. FIG. 20 shows results with HT-1080 cells and an ACPP cleavable by MMP-2. The intact peptide showed 18-fold less uptake than the equimolar mixture of the two fragments resulting from linker cleavage, which in turn was similar to a control CPP with only the polycation. The flow cytometric histograms showed that fluorescence on or in healthy cells was unimodal and reasonably homogeneous (FIG. 20A). Single cell microscopy (FIG. 20B) confirmed that cargo uptake was far greater after linker cleavage and indicated that a significant fraction reached the nucleus, as judged by accumulation of fluorescence in the nucleoli, similar to results previously reported for polycation-mediated transduction (17). Analogous cleavage-dependent association with cells was observed with a variety of ACPPs containing different numbers of arginine residues, different polyanionic sequences, and linkers cleavable by a variety of proteases, including enterokinase, MMP-2, MMP-9, and urokinase plasminogen activator, or even by simple reduction of a disulfide bond (Table 1). In the best case, cell labeling increased >100-fold when the polyanion was cut off from the polycation. Both the arginine residues and the acidic residues could be D-amino acids, as desirable to restrict in vivo proteolysis to the central linker between the two domains. Greater contrast was obtained when the polycationic, not the polyanionic, region was closer to the C terminus. We hypothesize that this preference is because the new amino terminus created by proteolytic cleavage would reinforce the polycationic charge, whereas, if the polycation is at the N terminus, proteolysis would append a negatively charged carboxylate to the polycation. Cleavage-dependent contrast was equally observable with fluorescein or the far-red fluorophore Cy5 as cargo and with or without a PEG tail (Table 1). Such PEGylation increases solubility and slows in vivo excretion but is not necessary to block CPP activity.

TABLE 1 Effect of different linkers and acidic inhibitory domains on ACPP association with Jurkat and HT-1030 cells assayed by flow cytometry Uptake Increase Uptake after caused before cleavage by Sequence cleavage (reagent) cleavage EEEEEDDDDK*AXRRRRRRRRRXC(F1) 0.18 2.4  13 SEQ ID NO: 52 (EK) EEEEEDDDDK*ARRRRRRRRRXC(F1) 0.07 1.2  17 SEQ ID NO: 53 (EK) EDDDDK*AXRRRRRRRRRXC(F1) 0.30 2.3   8 SEQ ID NO: 54 (EK) EEDDDDK*ARXRRXRRXRRXRRXC(F1) 0.015 0.11   7 SEQ ID NO: 55 (EK) DDDDDDK*ARRRRRRRRRXC(F1) 0.05 0.77  16 SEQ ID NO: 56 (EK) EEDDDDK*AXrrrrrrrrrXC(F1) 0.07 1.2  17 SEQ ID NO: 57 (EK) eeeeeeXPLG*LAGrrrrrrrrrXc(F1) 0.086, 1.3, 1.3  16, 39 (SEQ ID NO: 58) 10 min 0.034 (MMP-2) eeeeeeXPLG*LAGrrrrrrrrrXc(F1) 0.11 2.1  19 (SEQ ID NO: 58) 60 min (MMP-2) UeeeeeeeeXPLG*LAGrrrrrrrrrXk(F1) 0.006 0.74 123 SEQ ID NO: 59 (MMP-2) eeeeeeXPLG*LAGrrrrrrrrrXc(Cy5) nc nc  36 SEQ ID NO: 60 (MMP-2) UeeeeeeXPLG*LAGrrrrrrrrrXc(Cy5) nc nc  20 SEQ ID NO: 61 (MMP-2) UeeeeeeeeXPLG*LAGrrrrrrrrrXk(Cy5.5) nc nc  17 SEQ ID NO: 62 (MMP-2) [11-kDa PEG]XeeeeeeeeeXPLG*LAGrrrrrrrrrXk(Cy5) 0.012, 0.82, 2.1  68, 21 SEQ ID NO: 63 0.10 (MMP-2) 0.10 1.87  18 (MMP-9) [11-kDa PEG]XeeeeeeeeeXLALGPGrrrrrrrrrXk(Cy5)† 0.019 0.021   1.13 SEQ ID NO: 64 (MMP-2) — 0.020   1.07 (MMP-9) F1-XrrrrrrrrrXPLG*LAGeeeeeeee-βAla 0.004 0.06  16 SEQ ID NO: 65 (MMP-2) F1-XrrrrrrrrrXSGRS*Aeeeeeeee-βAla 0.012 0.05   4 SEQ ID NO: 66 (uPA) eeeeeeXSGRS*AXrrrrrrrrrXc(Cy5) nc nc  11 SEQ ID NO: 66 (uPA) Fl-rrrrrrrrr-*-ceeeeee‡ 0.092 0.72   8 SEQ ID NO: 67 (reduction) For sequences, lowercase characters indicate D-amino acid. All peptides were amidated at C terminus. Values represent the results of triplicate experiments performed on the same day. Some entries have two values because the triplicate experiments were repeated on another day. *Cleavage site; U, succinoyl; X, 6-aminohexanoyl; F1, fluorescein. Uptake before and after cleavage was measured by FACS, normalized to Fl-GGRRRRRRRRRR (SEQ ID NO: 68) or rrrrrrrrrk(Cy5) (SEQ ID NO: 69), except for some measurements not calibrated (nc) with respect to either reference peptide. EK, enterokinase; uPA, urokinase plasminogen activator. †This scrambled control should be uncleavable, so the rightmost column refers to increase due to enzyme exposure rather than cleavage. ‡Disulfide-linked. In “ceeeeee,” the N terminus is D-glu and the amidated C terminus is D-cys.

ACPPs Adopt a Hairpin Conformation Before Cleavage. Polyanion inhibition of polycation uptake would be most easily understood if the oppositely charged segments zippered together as shown in FIG. 19. Direct evidence for such a hairpin structure was obtained by homonuclear two-dimensional NMR analysis (see supporting information for methods). FIG. 21 shows nuclear Overhauser effects observed in two-dimensional NMR of a simple ACPP, succinyl-e₈-XPLGLAG-r₉-Xk, where X denotes 6-aminohexanoyl. (The cleavable peptide was [11 kDa PEG]X-e₉-XPLG*LAG-r₉ and the scrambled peptide was [11 kDaPEG]-X-e₉-XLALGPG-r₉). The observed nuclear Overhauser effect correlations shown in FIG. 21 reflect proton-proton proximities. Dashed red lines indicate observed nuclear Overhauser effects, and the green line highlights the peptide outline for clarity. The observed short-range couplings within the PLGLAG (SEQ ID NO: 1) linker indicate a turn conformation (see Example 13 for more detail). In addition, the numerous nuclear Overhauser effects between the strings of D-arginine and D-glutamate clearly indicate pairings that would stabilize the hairpin turn. Taken together, the data indicate that the presence of a hairpin structure, although they are not sufficient to define a complete atomic-level structure owing to chemical shift overlap.

MMP-2 Cleavable ACPPs Concentrate in Human Tumors Xenografted into Mice. We next tested whether ACPPs could light up proteaseexpressing human tumor xenografts in whole mice. We chose HT-1080 tumors in the axilla of nude mice because these tumors express both MMP-2 and MMP-9 and have been used to test other MMP-2 cleavable contrast agents (22, 26). Adding a PEG tail to the peptide proved helpful to prevent excessively rapid excretion; PEGs of ≈5, 11, and 21 kDa gave plasma half-lives of 4, 15, and 38 min, respectively, consistent with trends reported in ref. 27. Anesthetized mice were injected through the tail vein with either an MMP-2 cleavable ACPP, an isomeric scrambled version verified not to be a substrate for MMP-2 or MMP-9, or an all-D-amino acid version. All peptides had Cy5 attached to permit in vivo imaging of the far-red fluorescence through the skin. FIG. 22A1 shows that the tumor is the brightest fluorescence visible in the live animal injected with the MMP-2-cleavable ACPP, whereas FIG. 22B1 shows much less tumor contrast from a different animal injected with the scrambled analog. Similar cleavage-dependent contrast was seen in frozen sections at higher magnifications (FIGS. 22 A2, A3, B2, and B3). To quantitate the results, we measured the contrast index defined as (fluorescence intensity of tumor−autofluorescence)/(fluorescence of normal contralateral region−autofluorescence). This index was 2.1±0.17 (mean±SE, n=6) for the cleavable ACPP, which was modestly but significantly higher (P<0.02, two-tailed t test) than the values obtained for both the scrambled isomer (1.3±0.16, n=2) and the all-D-amino acid control (1.5±0.11, n=4). The latter values may differ from 1.0 because of the phenomenon of enhanced permeability and retention, whereby macromolecules passively accumulate in tumors because their vasculature is leakier than that of healthy tissue (28). Nevertheless, the amount of cleavable ACPP that accumulates in the tumor is significantly more than can be accounted for by the enhanced-permeability-and-retention effect, arguing for local unmasking of the CPP by enzymes secreted by the tumor.

Although FIG. 22A1 shows that tumors become visible in intact live animals, such fluorescence images are highly biased in favor of superficial tissues, skin>s.c. tumors>deep organs. To measure the true distribution of the peptides unbiased by anatomical depth, postmortem tissue samples from different organs were homogenized in detergent to release the labeled probe, clarified by centrifugation, and quantified by Cy5 fluorescence relative to tissue standards spiked with known amounts of dye. Standardized uptake values (SUVs), defined as (moles of recovered peptide/weight of tissue sample)/(moles injected into animal/total body weight), are shown in FIG. 23 as SUVs (mean±SD) 1 hr after injection of peptide into mice, comparing a cleavable ACPP with its all-D-amino acid control. The data shown in FIG. 23 are fluorescence measurements from solubilized tissue. The cleavable peptide was [11-kDa PEG]-X-e₉-XPLG*LAG-r₉-Xk(Cy5), and the uncleavable peptide was [11-kDa PEG]-X-e₉-Xplglag-r₉-Xk(Cy5). Although the kidney and liver have the highest absolute SUVs, as typical for peptides, the tumors gave a higher ratio of SUVs between the cleavable and control peptide: 3.1. Also, of the tissues with appreciable uptake, only in the tumors did the difference between the two peptides attain statistical significance (P<0.05, two-tailed t test). The standard deviation for the cleavable peptide was <0.05 for muscle, brain, and spleen and <0.05 for muscle, spleen, heart and pancreas for the uncleavable peptide.

ACPPs Light Up Human Squamous Cell Carcinomas. Although human tumor cell lines xenografted into immunodeficient mice are popular cancer models, they fail to mimic many aspects of real human tumors. To get a preliminary indication whether ACPPs would work on clinically relevant neoplasms, we applied ACPPs to coarse sections cut from tissue freshly resected from patients undergoing surgery for squamous cell carcinoma of the aerodigestive tract. These surgical samples contained both neoplastic and normal tissue, distinguishable by cell morphology and histological staining. The ACPP, whose covalently attached PEG was reduced to 5 kDa to facilitate diffusion, consistently stained tumor tissue more brightly than normal tissue, whereas the scrambled peptide or the ACPP coadministered with N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (a Zn²⁺ chelator and broad-spectrum MMP inhibitor), showed no such consistent pattern. In FIG. 23, A Upper-D Upper are Cy5 fluorescence images displayed at a uniform gain, whereas A Lower-D Lower are transmitted light views of the same fields. The squamous cell carcinoma tumor tissue exposed to cleavable peptide (FIG. 23A) was much more fluorescent than normal tissue exposed to cleavable peptide (FIG. 23B) or either tissue exposed to scrambled peptide (FIGS. 23 C and D). Contrast, defined as (tumor tissue fluorescence−autofluorescence)/(normal tissue fluorescence−autofluorescence), was almost eight in this example. Contrast tended to be greatest where the tumor tissue had a high histologic grade of malignancy. An example in FIG. 23A is that the keratin pearl, characteristic of differentiated squamous epithelium (29), was less fluorescent than the surrounding tumor. The contrast averaged 2.7±0.2 (mean±SD) from two patients with relatively differentiated oral cavity_oropharynx tumors (low to moderate histologic grade of malignancy), whereas two high-grade laryngeal tumors gave more contrast, 6.5±3.4. Also, lymphocytic granulation tissue was nearly as bright as the tumors themselves, possibly because of the release of MMPs from lymphocytes. Normal tissue immediately adjacent to tumor tissue was noticeably brighter than more remote normal tissue, possibly because of the presence of immune cells or to diffusion of the soluble proteases.

Discussion

We believe the selective activation of CPPs as disclosed herein offers many advantages, including the following advantages: (i) It should be adaptable to a wide variety of imaging and therapeutic modalities, including radioactivity, because the payload or cargo need not have any particular spectroscopic properties. CPP-mediated uptake has already been demonstrated with gamma-ray emitters and MRI contrast agents as well as potential therapeutic agents (30). Close integration between imaging and therapy would thus be facilitated; for example, providing ACPPs having nonoptical cargoes is a useful application of the present methods and compositions. (ii) Catalytic amplification is inherent in the methods disclose dherein; i.e., each protease molecule can cleave multiple substrate molecules, whereas with antibodies, for example, each epitope can only bind one antibody at a time. (iii) ACPPs help deliver the cargo not just to the surface of the target cell but inside and to the nucleus, which is important for therapeutic payloads and other payloads and applications. (iv) Molecular masses can be varied over a wide range from quite small (≈18 aa or ≈2 kDa, where “aa” indicates amino acid(s) and “kDa” indicates kiloDaltons) up to nanoparticles of several nanometers in diameter (15, 16, 31). Depending on whether polymers are appended to the polyanionic versus polycationic portion, one can choose whether they are discarded or retained after linker cleavage. Excessive molecular mass typically has the disadvantage of decreasing penetration into solid tumors, particularly when they have high interstitial fluid pressure (29). (v) The highly modular substrates are synthesized by standard methods of peptide synthesis and bioconjugation, without requiring fermentation or high-level expression systems, yet they contain enormous scope for rational or combinatorial variation. (vi) The high content of D-amino acids would be expected to reduce immunogenicity. Other guanidinium decorated nonpeptidic backbones, such as carbamates and peptoids, are known to be competent for cell uptake (32) and may be modulatable in analogy to the peptides discussed above. (vii) Extracellular proteases are mechanistically important in cancer (33), particularly in angiogenesis and metastasis, unlike many tumor antigens of unknown function. In principle, tumor cells that try to become resistant by down-regulating their proteases are likely to become less aggressive and metastatic. In addition, it is believed that multiple subtypes of cancers may share similar properties of up-regulating a relatively limited repertoire of proteases, giving each successful substrate a wider range of clinical indications. (viii) Proteases that are or can become extracellular are crucial to many other disease processes, including thrombosis, congestive heart failure, inflammation, neurodegeneration, and infectious pathogens (34-37). Uses of the methods, compositions and systems disclosed herein are not limited to proteases: any conditions that sever the vetoing polyanion from the polycation (e.g., agents that reduce disulfide bonds) may be used and exploited as mechanisms for localization.

The present examples in vivo have included substrates for soluble proteases, such as MMP-2 and MMP-9, mainly because these MMPs have well established roles in metastasis and angiogenesis, clear substrate preferences, and commercial sources for in vitro testing. However, soluble proteases may have the potential disadvantage of gradually leaking from the tumor into the general circulation, where they would contribute to background signal and reduced contrast. MMPs have been detected in the plasma and urine of cancer patients at levels that show positive correlations with the severity of metastatic disease (23), although the relative enzyme activities in tumors versus blood do not seem to be known. To circumvent diffusion of soluble MMPs, substrates for membrane-bound MMPs, such as MT1-MMP (24, 25) may be used. Other membrane-bound proteases including members of the ADAM (a disintegrin and metalloprotease) family (38) are also suitable alternatives to soluble MMPs and substrates for these and other proteases may be used in the practice of the methods disclosed herein.

Although the examples disclosed herein have not included incorporation of additional contrast mechanisms, such as fluorescence dequenching (22, 26) or enhanced permeability and retention of adequately large polymers (28), within tumors with leaky vasculature, such additional contrast mechanisms may be used in or with these methods. For example, if maximum contrast and sensitivity are desired, attachment of ACPPs to nanoparticles or large polymers may be done in order to harness the enhanced permeability-and-retention effect of such cargoes. In addition, for example, in the case of fluorescence, crowding fluorophores together on a polymer or nanoparticle (22, 26, 39, 40) or including a quencher on the end of the polyanion may be used to improve contrast by suppressing fluorescence of the uncleaved substrate.

Far-red fluorescence was used as an imaging modality in the examples, and offers at least the following advantages: the cyanine dyes are stable and easy to conjugate, the imaging equipment is relatively simple to use and cheap, and its spatial resolution spans the full range from subcellular to whole animal. In mice, fluorescence imaging can reach a significant fraction of the intact animal, especially when aided by tomographic techniques (40). In larger animals and in patients, the few-millimeters-deep penetration may restrict the utility of fluorescence to (i) the most superficial dermatological tumors, (ii) the retina, (iii) tumors near the surface of a body cavity accessible by endoscopy (41), and (iv) the margins of a surgical resection. An exemplary use of the present methods is the real-time molecular imaging of the margins of a resection while the patient is still on the operating table. Such a use would be of great value to the surgeon to decide whether any invasive carcinoma tissue remained lurking at or just beyond the tissue just removed. Instrumentation for infrared image-guided surgery has been described (42) and may be useful for such methods. Contrast agents comprising compositions as described herein may be provided to target tissue by topical application, by intravenous infusion, or other means.

The ability of a polyanionic peptide domain to inhibit binding and entry of a closely apposed polycationic CPP is functionally reminiscent of intramolecular fluorescence resonance energy transfer, in which an acceptor chromophore quenches the fluorescence of a nearby donor fluorophore. In each case, if the linker is cleaved and the inhibitory moiety diffuses away, the active partner (the CPP or the donor fluorophore) is unmasked. The unmasking of CPPs has a completely different underlying mechanism and a much slower time scale than intramolecular fluorescence resonance energy transfer, but offers a much broader range of useful imaging modalities and cargoes than does fluorescence resonance energy transfer.

REFERENCES FOR EXAMPLE 12

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Example 13

Further Exemplary Material Regarding Tumor Imaging Via Proteolytic Activation of Cell Penetrating Peptides

Reagents: Fmoc protected amino acids and synthesis resins were purchased from EMD Chemicals Inc. Dimethylformamide (DMF), piperidine, and 2-(1H-9-azabenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) were from Applied Biosystems. Trifluoroacetic acid (TFA), thioanisole, triisopropylsilane, ethanedithiol, and diisopropylethylamine (DIEA) were from Sigma-Aldrich. 5(6)-Carboxyfluorescein succinimidyl ester and 5-iodoacetamidofluorescein were from Molecular Probes. Cy5 monoreactive NHS ester and Cy5 monomaleimide were from Amersham Biosciences. MMP-2 proenzyme and MMP-9 were from EMD. Enterokinase and urokinase plasminogen activator (uPA) were from Invitrogen and Alexis, respectively. Methoxy PEG-maleimide (5 KDa, 21 KDa) was purchased from Nektar and methoxy PEGmaleimide (11 KDa) was supplied by SunBio PEG-SHOP, Korea. All reagents were used as obtained without further purification.

Peptide Synthesis and Fluorophore Labeling: Peptides were synthesized on an automatic peptide synthesizer (Pioneer Peptide Synthesis System by Applied Biosystems) using standard protocols for Fmoc solid phase synthesis. After the peptide was synthesized, the resin was washed with dimethylformamide, dichloromethane, and methanol 3 times each and vacuum dried for 3 hr. The peptides were cleaved off the resin overnight with either CF3COOH/thioanisole/triisopropylsilane (96/2/2, v/v) for peptides without a sulfhydryl group, or CF3COOH/thioanisole/triisopropylsilane/ethanedithiol (94/2/2/2, v/v) for peptides with a sulfhydryl group. The cleavage solution was evaporated nearly to dryness, and the crude peptide was triturated with ether and vacuum dried for 3 hr. Fluorophores were attached to peptides either before or after cleavage from the resin; 5(6)-carboxyfluorescein Nhydroxysuccinimidyl ester and Cy5 monoreactive N-hydroxysuccinimidyl ester labeled amino groups, whereas 5-iodoacetamidofluorescein and Cy5 monomaleimide reacted with sulfhydryl groups. Finally, fluorophore labeled peptides were purified on HPLC (C18 reverse phase column, eluted with 10-40% acetonitrile in water with 0.1% CF3COOH) and lyophilized overnight. The molecular weight of all peptides was confirmed by mass spectroscopy, and the concentration of each peptide stock solution was verified by UVvis absorbance.

Peptide disulfide bond formation and reduction: Peptides with cysteine residues were cleaved off the resin via standard procedures. To form a cyclic disulfide, vacuum dried crude peptide was diluted to 1 mg/ml in 5 mM NH4HCO3 and vigorously stirred in air for 3 hr. The crude cyclic peptide was purified on HPLC(C18 reverse phase column, eluted with 10-40% acetonitrile in water with 0.1% TFA) and lyophilized overnight. As before, the molecular weight of each peptide was confirmed by mass spectroscopy, and the concentration of the stock solution was verified by UV-vis absorbance. To obtain the linear peptide, the disulfide bond was reduced by mixing equal volumes of 100 μM cyclic peptide, 10 mM TCEP [tris(2-carboxyethyl)phosphine], and 100 mM MES [2-mercaptoethanesulfonic acid, sodium salt] in PBS and incubating at room temperature for 30 min. Reduction was confirmed by HPLC and mass spectroscopy. The final concentrations of TCEP and MES in the media during cell uptake assays were 0.5 and 5 mM respectively.

PEGylated peptide synthesis and Cy5 labeling: Peptides with free thiol groups at the N-terminus were synthesized using a standard Fmoc peptide synthesis protocol, except that the final amino acid coupled to the resin was tritylmercaptoacetic acid. The peptide was cleaved off the resin through the standard procedure described earlier and reacted with 0.5-0.8 equivalent methoxy PEGmaleimide in DMF and 100-fold excess 4-methymorpholine as base at room temperature for over 12 hours. Solvent and excess base were evaporated off under vacuum. The pegylated peptide was labeled with Cy5 by reacting with 2-3 equivalent Cy5 mono NHS ester in 50 mM sodium bicarbonate solution at room temperature overnight. The crude product was purified on HPLC and then lyophilized.

ACPP Cleavage by Enterokinase: 10 μl of a 0.38 mM peptide stock solution dissolved in water was mixed with 10 μl 1 U/μl enterokinase (Invitrogen) and incubated at 37° C. Enzymatic cleavage was monitored by injecting 5 μl of the reaction mixture on HPLC and observing either UVvis absorbance at 440 nm for fluorescein labeled or 650 nm for Cy5 labeled peptide. The HPLC chromatograms showed that cleavage by enterokinase was nearly complete after 15 min incubation time. The new peaks were collected and their identities were determined by mass spectroscopy. The mass spectra indicated that the enzyme cut between lysine and alanine residues of the enterokinase substrates as expected.

ACPP cleaved by urokinase plasminogen activator (uPA): 100 μM peptide in 400 μA PBS (Phosphate Buffered Saline, pH7.4) was incubated at 37° C. with 6 μg uPA for over 3 hours. The cleavage progress was monitored on HPLC. Mass spectroscopy on HPLC fractions indicated that the cleavage was close to completion after 3 hr and that the enzyme cleavage site was between arginine and serine residues in the peptide as expected (1) (MS: 2688.6 found, 2688.2 calculated).

Conformational analysis by two-dimensional NMR: We have studied the peptide using homonuclear two-dimensional NMR spectra in order to assess structural proclivities of the native ensemble. The NMR samples were prepared in 90% H2O, 10% D2O buffer containing 50 mM potassium phosphate, pH 6.5. Peptide concentration was 2.69 mM and spectra were recorded at 5° C. NMR spectra were collected using a Bruker DMX 500 MHz spectrometer and a Varian UnityPlus 800 MHz spectrometer. DQF-COSY, TOCSY, and NOESY spectra were collected using standard pulse sequences (see (2) and references therein). All spectra were collected using the 3-9-19 pulse sequence with gradients for water suppression (3). The NOE mixing time was 500 ins and the TOCSY mixing time was 60 ms.

Spectral processing was performed using Felix (Molecular Simulations Inc., San Diego, Calif.). Apodizations in the t2 and t1 dimensions were with cosine squared window functions and the solvent was deconvoluted from the spectra using the time domain convolution method (4) with a sine bell function.

As expected, there is chemical shift degeneracy and spectral overlap introduced by the strings of D-glu and D-arg residues (DQF-COSY data not shown). We can classify the resonances of the D-glu and D-arg residues by type and make sequence specific assignments of the resonances of linker region, i.e. XPLGLAG (SEQ ID NO: 70). In FIGS. 24 and 25, the consistency of the observed NOEs are assessed relative to the sequential and medium-range NOEs expected for a B-turn and the long-range NOES for cross-strand interactions.

FIG. 24A shows the H^(β)/H^(γ)/sidechain (δ₁)-H^(N)(δ₂) region of the NOESY spectrum, and FIG. 24B shows the identical region for the TOCSY spectrum. The H^(β) and H^(γ) shifts of the D-glu and D-arg resonances are labeled in the TOCSY. Significant chemical shift overlap is present among H^(β) and H^(γ) resonances, but resonances by amino acid types are well resolved. In both FIGS. 24A and 24B, there is a cluster of H^(β) and H^(γ) shifts at 1.7/1.78 and 1.55/1.66 respectively for D-arg and H^(β) and H^(γ) shifts at 1.95/2.03 and 2.25/2.3 respectively for D-glu. In the NOESY spectrum (FIG. 24A), we see evidence for crossstrand interactions between the string of D-arg and the string of D-glu.residues. NOE cross-peaks at 1.92, 2.03, 2.25, and 2.29 (δ₁) and 8.65 (δ₂) are consistent with through space interactions between the H^(β) and H^(γ) sidechain of one or more D-glu and the H^(N) backbone of one or more D-arg.

For clarity, 1D vectors drawn from selected D-arg and D-glu backbone H^(N) resonances (δ₂) (see dashed blue lines in FIGS. 24A and 24B) are depicted in FIG. 24C-24E. FIG. 24C is drawn from the NOESY spectrum at 8.65 ppm (δ₂), the H^(N) resonance of a D-arg, with H^(β) and H^(γ) signals of both D-glu and D-arg present. FIG. 24D is drawn from the TOCSY spectrum at the same δ₂ shift and H^(N) resonance, with only the H^(β) and H^(γ) signals of D-arg. FIG. 24D is also drawn from the TOCSY spectrum but 8.59 ppm (δ₂), the H^(N) resonance of a D-glu, with only the H^(β) and H^(γ) signals of D-glu.

FIG. 25 shows the H^(N) (δ₁)-H^(N)(δ₂) region of the NOESY spectrum. There are five cross-peaks labeled with sequence-specific identification, indicating sequential H^(N)-H^(N) backbone interactions among the residues of the linker region and the neighboring D-glu and D-arg on either side. This type of short-range NOE is consistent with turn or helical secondary structure (5).

Potential interactions with furin: Up to now, we have relied almost exclusively on 9 arginines, usually D-, in a row, partly for simplicity and partly because they are amongst the most effective uptake sequences (6, 7). However, very recently nona-D-arginine amide has been reported to be a potent inhibitor of furin, a well-known processing protease (8). Given the highly electrostatic nature of the binding, it is quite likely that the intact substrate with polyanionic domain still attached will be a much poorer inhibitor of furin. If this prediction is verified experimentally, then the furin inhibition may be unimportant or beneficial, because it will be mainly in target tissue such as tumors that furin is acutely inhibited.

Imaging of SCCA samples with ACPP's following administration of an MMP inhibitor: Since MMP's are dependent on zinc for activation, we used the lipid soluble, high affinity Zn²⁺ chelator TPEN (N,N,N′,N′-tetrakis-(2-pyridylmethyl)ethylenediamine) (9) as a broad spectrum MMP inhibitor to preliminarily assess whether cleavage and retention of our peptide in SCCA tumors was MMP dependent. Fresh SCCA slices were incubated in HBSS (FIG. 26A) or 1 μM TPEN in HBSS (FIG. 26B) at room temperature for 15 minutes. Slices were then stained with 1 μM cleavable peptide alone (FIG. 26A) or 1 μM cleavable peptide plus 1 μM TPEN (FIG. 26B) before being washed five times in fresh HBSS and cryosectioned. The images shown in FIGS. 26A and 26B were taken using a 10× objective, and hematoxylin/eosin staining was used to verify tissue type.

REFERENCE LIST FOR EXAMPLE 13

-   1. Ke, S. H., Coombs, G. S., Tachias, K., Corey, D. R. &     Madison, E. L. (1997) J. Biol. Chem. 272, 20456-20462. -   2. Ernst, R. R., Bodenhausen, G. & Wokaun, A. (1990) Principles of     Nuclear Magnetic Resonance in One and Two Dimensions (Oxford     University Press, Oxford). -   3. Piotto, M., Saudek, V. & Sklenar, V. (1992) J. Biomol. NMR 2,     661-665. -   4. Marion, D., Ikura, M. & Bax, A. (2004) J. Magn. Reson. 84,     425-430. -   5. Wiithrich, K. (1986) NMR of Proteins and Nucleic Acids (John     Wiley & Sons, New York). -   6. Mitchell, D. J., Kim, D. T., Steinman, L., Fathman, C. G. &     Rothbard, J. B. (2000) J. Peptide Res. 56, 318-325. -   7. Gammon, S. T., Villalobos, V. M., Prior, J. L., Sharma, V. &     Piwnica-Worms, D. (2003) Bioconjugate Chem. 14, 368-376. -   8. Kacprzak, M. M., Peinado, J. R., Than, M. E., Appel, J., Henrich,     S., Lipkind, G., Houghten, R. A., Bode, W. & Lindberg, I. (2004) J.     Biol. Chem. 279, 36788-36794. -   9. Arslan, P., Di Virgilio, F., Beltrame, M., Tsien, R. Y. &     Pozzan, T. (1985) J. Biol. Chem. 260, 2719-2727.

Example 14

Cleavage kinetics for MMP-2 cleavage of H₂N-e₆-XPLGLAG-r₉-Xc(Cy5)-CONH₂ (where X ≡aminohexanoic acid) are illustrated in FIG. 27. As shown in this figure, the K_(m) for this cleavage is 534 μM; the k_(cat) is 15.0 s⁻¹ and the ratio k_(cat)/K_(m) is 28,037 M⁻¹ s⁻¹.

Example 15

Cleavage of a MMP-2 substrate ACPP is illustrated in FIG. 27. The kinetics of cleavage of the ACPP peptide H₂N-e₆-XPLGLAG-r₉-Xc(Cy5)-CONH₂ (SEQ ID NO: 71) (where (X ≡aminohexanoic acid, also termed aminocaproic acid) have a K_(m) of 534 μM, a k_(cat) of 15.0 s⁻¹ and a k_(cat)/K_(m) of 28,037 M⁻¹ s⁻¹. As shown in FIG. 28, uptake into live, unfixed cells is dependent on enzymatic cleavage of the ACPP peptide by MMP-2. The cleavage site is between the first G and the second L in the sequence PLGLAG, as indicated in the figure. The ACPP peptides may be labeled with Cy5 dye (SEQ ID NO: 71), with fluorescein (Fluor) (e.g., H₂N-eeeeee-(ahx)-PLG LAG-rrrrrrrrr-(ahx)-c(Fluor)-CONH₂ (SEQ ID NO: 72), or with other labels. FIG. 29 shows fluorescence images from HT-1080 cells treated with the Cy5-labeled ACPP peptide XeeeeeeeeeXPLGLAGrrrrrrrrrXk (SEQ ID NO: 52) that has been PEGylated with an 11 kDa PEG moiety. The images shown in FIG. 29 demonstrate increased uptake of the cleaved peptide compared to the uncleaved peptide, and demonstrate localization of the cleaved peptide to the nucleus of these cells as well as to cytoplasmic compartments.

Example 16

Images taken from nude mice bearing MMP-2-positive tumors demonstrate that cleavage leads to uptake and localization of the ACPP peptide fragments in tumor tissue (FIG. 30). Control “scramble” peptides lacking a MMP-2 cleavage site (eeeeeeeeeXLALGPG-rrrrrrrrrXk(Cy5) (SEQ ID NO: 73) are not concentrated in tumor tissue, while fluorescence from cleavable peptides including a MMP-2 cleavage site is much higher in tumor tissue than in other tissues and higher than in tumor tissue of mice receiving the control peptides. Images were taken 17 min after injection of Cy-5-labeled peptides into tail vein. Note that bladder and salivary gland also fluoresce, and that the gut also shows some autofluorescence. As shown in FIG. 31, the greatest Cy5 fluorescence intensity was found over tumor tissue treated with cleavable peptides. Images shown in FIG. 32 show contrast enhancement by the cleaved peptides compared to the uncleaved peptides. Human HT-1080 tumors xenografted into mice are more readily discernable in live and in hiostological images with cleaved peptides than with uncleaved peptides. Similar images are shown in FIG. 33 showing that cleaved peptides improve imaging of spontaneous mammary tumors in MMTV-polyoma middle T, iNOS −/− mice. An image taken 55 min after tail vein injection into a mouse having a tumor shows significant intensity over a tumor. Histology of a similar tumor suggests accumulation in stromal annulus rather than tumor core. Gel images confirm that such tumors contain the cleaved peptide. The figure also indicates that uncleaved and uncleavable (all-D amino acid version) are not significantly taken up into tumors. As shown in FIG. 34, a PEGylated cleavable peptide with RGD labels tumor metastasis and labels surrounding macrophages in lymph nodes in MMTV-polyoma middle T, iNOS −/− mice. EXAMPLE 17

FIG. 35 shows human squamous cell carcinoma tissue resected from a patient, including adjacent normal tongue tissue as a control. Images from tumor tissue treated with cleavable peptide are much clearer than are images from similar tumor tissue treated with “scrambled” uncleavable peptides and are much clearer than images from normal tongue tissue treated with cleavable peptide. The tumor tissue contains much more gelatinase than normal tongue tissue. Similar images in FIG. 36 further demonstrate the utility of cleavable peptides for imaging of tumor tissue. Fresh tumor tissue was sliced in 1-mm slices and incubated in cleavable or uncleavable peptide for 15 min, washed, and frozen. Sections were taken for fluorescence microscopy using a low-power objective, and tissue type was verified by staining tissue. The arrow in the picture on the left indicates a differentiated keratin pearl. As a control, histologically normal tissue from the same patient was treated similarly with MMP-2 cleavable peptide or scrambled peptide. The arrow in the third figure form the left indicates tumor cells. EXAMPLE 18

Phage may be transported into cells by ACPP peptides. As shown in FIG. 37, which presents a scheme for coating Filamentous M13 phage with ACPPs, and FIG. 38, which provides further methods and shows increased uptake of coated phage in tumor tissue, phage particles may be coated with cleavable peptides for directed delivery into cells upon cleavage of the inactivating portion or the cleavable peptides. About 30-50 copies of the ACPP may be incorporated per phage, where as indicated in the example shown, the ACPP may be attached to pIII coat protein or other attachment moieties. Cleavable peptides may include, for example, PLGLAG, while the uncleavable peptides may include the scrambled peptide LALGPG. The masked M13 phage is one that includes the ACPP particles before cleavage. Upon enzymatic activation, the phage becomes activated, having a positively charged exterior, and becomes a tumor cell binding phage. M13 phage are indicated in FIG. 37 for coating and activation, and such activatable phage are shown to be taken up by tumors in FIG. 38. Sequence-dependent phage accumulation in xenografted tumors was demonstrated as shown in FIG. 38. Phage was injected into the tail vein of mice bearing xenografted tumors; after 3 hours, the organs and tumor xenograft were removed from the mice and ground up. The ground up tissue was added to bacteria. Bacteria were then plated for calculation of the resulting phage titer. Phage carrying cleavable peptides were much more readily taken up by tumors than were phage carrying uncleavable peptides. In addition, the background labeling of liver and kidney seems less severe with phage than with dye cargoes. It is not essential that the charged amino acids be D-amino acids. Thus, a selective uptake mechanism has been demonstrated and can work on cargo as large as phage, demonstrating that large phage libraries may be built to find optimal tumor-specific sequences.

Other phage may be used as well, including T7 phage, λ phage, P4 phage, T4 phage, MS2 phage, and others. For example, T7 phage type 10-3b, T7 phage type 415-1b as well as Pcomb type M13 phage may be used to provide coated, activatable phage for delivery of phage and other cargo to target cells and tissues. EXAMPLE 19

ACPPs may be used to deliver radioactive cargo to target cells and tissues. An ACPP may be linked with a radioactive moiety (either directly or indirectly, covalently or non-covalently. FIG. 39 provides a scheme for production of an MMP substrate with a ^(99m)Tc Chelator as a payload. As illustrated in FIG. 39, a PEGylated MMP substrate peptide mPEG(11 kd)-S—CH₂—CONH-ahx-e₉-ahx-PLGLAG-r₉-ahx-k-CONH₂ (SEQ ID NO: 74) may be linked with a radioactive technetium atom for use in a cell uptake assay. Such ACPPs were produced and tested for cargo-delivery efficacy with Jurkat cells. In the experiments summarized in FIG. 40, 2 μM peptide was incubated either intact or following cleavage by the matrix metalloprotease MMP-9. Spiked with 0.25 mCi Tc label, 25 nM intact or cleaved substrate. The target Jurkat cells were incubated with the peptides for 30 min at 37° C., and then washed 3 times with HBSS (HEPES-buffered saline solution) at room temperature (RT). 20 hours later, cells were counted by Gamma counter. As shown in FIG. 40, although little radioactivity was associated with the Jurkat cells before cleavage, after cleavage of the ACPP to activate cargo delivery, there was a 38-fold increase in radioactivity associated with the target cells.

Further experiments demonstrating delivery of radioactive cargo to target cells with a technetium chelating moiety is shown in FIG. 41. Spiked (4%) with 60 μCi ^(99m)Tc-labeled peptide, the target Jurkat cells were loaded with 2 μM intact or MMP-9-cleaved peptide at 37° C. for 30 min. Then, the cells were washed with RT HBSS 0.3 times, and counted (Gamma counter) 20 hours later. In the experiment illustrated in FIG. 41, cleavage of the ACPP resulted in a 57-fold increase in radioactivity associated with the target cells. The technetium chelating moieties used in these studies are shown in the Figures. These experiments demonstrate in particular that ^(99m)Tc can be selectively accumulated in target cells.

Example 20

ACPPs may be used to deliver contrast materials to target tissues and cells, such as materials that enhance contrast for imaging. An ACPP may be linked with a contrast-enhancing agent directly or indirectly, covalently or non-covalently. Such imaging may be by magnetic resonance (MRI), x-ray (e.g., computer assisted tomography (CAT)), positron emission tomography (PET), single photon emission computed tomography (SPECT), neutron computed tomography (NCT), ultrasound, near infra-red (NIR) imaging, or other imaging means or methods, as well as radiation sensitizers (¹⁰B, ¹⁵⁷Gd) and chemotherapeutic agents (e.g., doxorubicin) for therapy, research, identification, of target tissues and cells or other purposes. For example, these methods are cascadable with other contrast mechanisms such as EPR (enhanced permeability and retention), retention by intracellular enzymes, fluorescence dequenching, rotational immobilization. ¹⁵⁷Gd is particularly attractive for combined MRI and NCT. FIG. 42 provides a scheme for production of an MMP substrate with an attached contrast enhancing agent (in this example, gadolinium). Gadolinium chloride (GdCl₃) is used to add gadolinium (Gd) to provide a contrast-enhancing, PEGylated ACPP mPEG-S—CH₂CONH-e₉-ahx-PLGLAG-r₉-K[DOTA(Gd)]-CONH₂ (SEQ ID NO: 75). It will be understood that in other embodiments the ACPP need not include a PEG moiety, and may include other moieties as well. As shown in FIG. 43, cleavage of the ACPP activates the uptake of MRI imaging reagent to nearly mM concentrations. Jurkat cells were incubated with 8 μM of the intact or of the cleaved substrate at 37° C. for 30 min; then the cells were washed four times with RT HBSS. Gd concentration was determined by ICP-MS (inductively coupled plasma mass spectroscopy). Cleavage led to a 19-fold increase in uptake by target cells.

Example 21

yclic Substrate for MMPs is illustrated in FIG. 44. A cyclic ACPP molecule can be linked to a cargo, PEGylated, or otherwise modified as indicated in the figure. Cyclic ACPP molecules offer the advantages of requiring cleavage at two sites to activate cell-uptake (e.g., enzymatic cleavage by MMP as illustrated in FIG. 44). A requirement for cleavage at two sites is an advantage, since then cleavage will be more sensitive to enzymatic concentration than molecules with a single cleavage site. Such a requirement is useful to improve contrast. FIGS. 45 and 46 illustrate exemplary synthetic schemes for producing cyclic ACPPs. Note that these molecules may be PEGylated.

As indicated in FIG. 47, a further advantage of cyclic ACPPs is that they may be self-quenching. As shown in FIG. 47, fluorescence from the cyclic ACPP shown in the example increases upon enzymatic cleavage. As shown in FIGS. 48-50, in vivo experiments demonstrate uptake, accumulation, cleavage and excretion of such peptides by liver and kidney. Thus, such molecules may be administered in vivo, and are cleaved in vivo.

FIG. 48 further illustrates ACPPs that require enzymatic action at multiple sites, including cyclic ACPPs and branched ACPPs. Where both of the cleavage sites are substrates for a single enzyme, the uptake should be proportional to the square of the protease concentration. However, with multiple enzymatic sites, an ACPP molecule may include cleavage sites that are substrates for more than one enzyme. Where an ACPP has two cleavage sites, each being a substrate of different enzymes, the uptake should be proportional to the product of the concentration of the first enzyme times the concentration of the second enzyme. ACPPs may include more than two cleavage sites, with uptake kinetics roughly following the number of cleavage sites (e.g., with three identical cleavage sites, the uptake should be approximately proportional to the cube of the enzyme concentration).

Thus, ACPPs may be linear, cyclic, branched or have other or mixed geometries. They may be used to deliver fluorescent, radioactive, or other labels, may deliver contrast agents, therapeutic agents, or multiple agents. Linkers can be cleaved by proteases, by reduction of disulfide bond, or by acidic or other conditions. Suitable enzymes and exemplary targets related to these enzymes include matrix metalloproteinases (for, e.g., cancer, stroke, and other conditions); Urokinase plasminogen activator (uPA) (for, e.g., cancer and other conditions); Prostate-specific antigen (for, e.g., cancer and other conditions); Thrombin and clotting cascade (for, e.g., thrombosis and other blood-related conditions); Reduction by leaked thiols under hypoxic conditions (for, e.g., cancer, infarcts and other conditions that lead to, or are caused by, hypoxic conditions); phosphatases (for, e.g., osteoporosis or other conditions); calpains (for, e.g., necrotic cells or other conditions); light (for, e.g., refining the specificity of photodynamic therapy and other uses).

Delivery of cargo by ACPPs provides advantages of other transport or therapeutic systems. Targeting is accomplished without the need for antibodies or antigens. ACPP targeting offers enzymatic amplification and transport of cargo into the nucleus of target cells. The ACPP peptides are relatively easy to synthesize and to vary combinatorially, so their ease of production offers advantages. Protease activities are mechanistically important and interesting, and the exploitation of such activities offers advantages including that the cleavage routes are well-studied and will continue to be studied and characterized. Membrane-bound proteases may be used to cleave ACPPs and to give higher contrast than soluble secreted proteases. The cleavable ACPPs are active in vivo. Some labeling of local bystander cells is expected and desirable. The cleavable ACPPs disclosed herein provide an extracellular analog of fluorescence resonance emission transfer (FRET) at least in that the anionic portions of ACPPs may serve to neutralize the cationic portions before cleavage. The ACPPs are applicable to all imaging modalities, including fluorescence and other imaging modalities. 

1. A cyclic molecule of the structure A-X1-B-X2, wherein B is a peptide portion of about 5 to about 20 basic amino acid residues, which is suitable for cellular uptake, A is a peptide portion of about 2 to about 20 acidic amino acid residues, which when linked with portion B is effective to inhibit or prevent cellular uptake of portion B; and A and B are linked together and linked via linkers X1 and X2 to form a cyclic compound; where X1 and X2 are each a cleavable linker of about 2 to about 100 atoms joining A with B to form a cyclic compound where each of X1 and X2 link to both A and B.
 2. The molecule of claim 1, wherein at least one of cleavable linkers X1 or X2 is configured for cleavage exterior to a cell.
 3. The molecule of claim 1, wherein at least one of cleavable linkers X1 or X2 is configured for cleavage interior to a cell.
 4. The molecule of claim 2, wherein at least one of cleavable linkers X1 or X2 is configured for cleavage by an enzyme.
 5. The molecule of claim 4, wherein said enzyme is a matrix metalloprotease.
 6. The molecule of claim 5 wherein at least one of cleavable linkers X1 or X2 comprises the amino acid sequence PLGLAG (SEQ ID NO:1).
 7. The molecule of claim 5 wherein at least one of cleavable linkers X1 or X2 comprises the amino acid sequence EDDDDKA (SEQ ID NO:2).
 8. The molecule of claim 1 wherein at least one of cleavable linkers X1 or X2 comprises a S-S linkage.
 9. The molecule of claim 1, further comprising a cargo moiety C linked to peptide portion B.
 10. The molecule of claim 9, wherein said cargo moiety C comprises a label.
 11. The molecule of claim 10, wherein said label comprises a label selected from the group consisting of fluorescent labels, luminescent labels, radioactive labels, contrast agents, radiation sensitizers, antibodies, and antigens.
 12. The molecule of claim 9, wherein said cargo moiety C comprises a phage.
 13. The molecule of claim 11, wherein said cargo moiety C comprises a radioactive technetium label.
 14. The molecule of claim 9, wherein said cargo moiety C comprises a contrast agent.
 15. The molecule of claim 14, wherein said cargo moiety C comprises a gadolinium contrast agent.
 16. A pharmaceutical composition comprising: A cyclic molecule of the structure A-X1-B-X2, wherein B is a peptide portion of about 5 to about 20 basic amino acid residues, which is suitable for cellular uptake, A is a peptide portion of about 2 to about 20 acidic amino acid residues, which when linked with portion B is effective to inhibit or prevent cellular uptake of portion B; and A and B are linked together and linked via linkers X1 and X2 to form a cyclic compound; where X1 and X2 are each a cleavable linker of about 2 to about 100 atoms joining A with B to form a cyclic compound where each of X1 and X2 link to both A and B. and a pharmaceutically acceptable carrier.
 17. The pharmaceutical composition of claim 16, further comprising a cargo moiety C linked to peptide portion B.
 18. The pharmaceutical composition of claim 17, wherein said cargo moiety C comprises a therapeutic agent.
 19. The pharmaceutical composition of claim 17, wherein said cargo moiety C comprises a radioactive agent.
 20. A method of modulating cellular uptake of a cargo moiety C, comprising: covalently attaching a cargo moiety C to a peptide B of about 5 to about 20 basic amino acid residues to form a molecule BC; linking said molecule BC to a peptide A of about 2 to about 20 acidic amino acid residues with cleavable linkers X1 and X2 of about 3 to about 30 atoms to form a cyclic molecule of the structure A-X1-BC-X2 where each of X1 and X2 link to both A and BC, and cleaving said cleavable linkers X1 and X2 effective to separate BC from said peptide A.
 21. A molecule for transporting a cargo moiety across a cell membrane of the structure A-X-B-C, wherein C is a portion comprising a cargo moiety, B is a peptide portion of about 5 to about 20 basic amino acid residues, which is suitable for cellular uptake, is covalently linked to portion C, and is effective to enhance transport of cargo portion C across a cell membrane, A is a peptide portion of about 2 to about 20 acidic amino acid residues, which when linked with portion B is effective to inhibit or prevent cellular uptake of B-C, and X is a cleavable linker of about 2 to about 100 atoms joining A with B-C, which can be cleaved under conditions in or near a target cell.
 22. The molecule for transporting a cargo moiety across a cell membrane of claim 21, wherein said portion comprising a cargo moiety C comprises a phage.
 23. The molecule of claim 21, wherein said portion comprising a cargo moiety C comprises a label.
 24. The molecule of claim 23, wherein said label comprises a label selected from the group consisting of fluorescent labels, luminescent labels, radioactive labels, contrast agents, radiation sensitizers, antibodies, and antigens.
 25. The molecule of claim 21, wherein said portion comprising a cargo moiety C comprises a radioactive technetium label.
 26. The molecule of claim 21, wherein said portion comprising a cargo moiety C comprises a contrast agent.
 27. The molecule of claim 21, wherein said portion comprising a cargo moiety C comprises a gadolinium contrast agent. 